Human coagulation factor VII polypeptides

ABSTRACT

The present invention relates to novel human coagulation Factor VIIa variants having coagulant activity as well as polynucleotide constructs encoding such variants, vectors and host cells comprising and expressing the polynucleotide, pharmaceutical compositions, uses and methods of treatment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. 119 of Danishapplication no. PA 2002 01423 filed Sep. 25, 2002 and U.S. applicationNo. 60/417,927 filed Oct. 11, 2002, the contents of which are fullyincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to novel human coagulation Factor VIIapolypeptides having coagulant activity as well as polynucleotideconstructs encoding such polypeptides, vectors and host cells comprisingand expressing the polynucleotide, pharmaceutical compositions, uses andmethods of treatment.

BACKGROUND OF THE INVENTION

Blood coagulation is a process consisting of a complex interaction ofvarious blood components (or factors) that eventually gives raise to afibrin clot. Generally, the blood components, which participate in whathas been referred to as the coagulation “cascade”, are enzymaticallyinactive proteins (proenzymes or zymogens) that are converted toproteolytic enzymes by the action of an activator (which itself is anactivated clotting factor). Coagulation factors that have undergone sucha conversion are generally referred to as “active factors”, and aredesignated by the addition of the letter “a” to the name of thecoagulation factor (e.g. Factor VIIa).

Initiation of the haemostatic process is mediated by the formation of acomplex between tissue factor, exposed as a result of injury to thevessel wall, and Factor VIIa. This complex then converts Factors IX andX to their active forms. Factor Xa converts limited amounts ofprothrombin to thrombin on the tissue factor-bearing cell. Thrombinactivates platelets and Factors V and VIII into Factors Va and VIIIa,both cofactors in the further process leading to the full thrombinburst. This process includes generation of Factor Xa by Factor IXa (incomplex with factor VIIIa) and occurs on the surface of activatedplatelets. Thrombin finally converts fibrinogen to fibrin resulting information of a fibrin clot. In recent years Factor VII and tissue factorhave been found to be the main initiators of blood coagulation.

Factor VII is a trace plasma glycoprotein that circulates in blood as asingle-chain zymogen. The zymogen is catalytically inactive.Single-chain Factor VII may be converted to two-chain Factor VIIa byFactor Xa, Factor XIIa, Factor IXa, Factor VIIa or thrombin in vitro.Factor Xa is believed to be the major physiological activator of FactorVII. Like several other plasma proteins involved in haemostasis, FactorVII is dependent on Vitamin K for its activity, which is required forthe gamma-carboxylation of multiple glutamic acid residues that areclustered close to the amino terminus of the protein. Thesegamma-carboxylated glutamic acids are required for the metal ion-inducedinteraction of Factor VII with phospholipids. The conversion of zymogenFactor VII into the activated two-chain molecule occurs by cleavage ofan internal Arg₁₅₂—Ile₁₅₃ peptide bond. In the presence of tissuefactor, phospholipids and calcium ions, the two-chain Factor VIIarapidly activates Factor X or Factor IX by limited proteolysis.

It is often desirable to stimulate or improve the coagulation cascade ina subject. Factor VIIa has been used to control bleeding disorders thathave several causes such as clotting factor deficiencies (e.g.haemophilia A and B or deficiency of coagulation Factors Xl or VII) orclotting factor inhibitors. Factor VIIa has also been used to controlexcessive bleeding occurring in subjects with a normally functioningblood clotting cascade (no clotting factor deficiencies or inhibitorsagainst any of the coagulation factors). Such bleeding may, for example,be caused by a defective platelet function, thrombocytopenia or vonWillebrand's disease. Bleeding is also a major problem in connectionwith surgery and other forms of tissue damage.

European Patent No. 200,421 (ZymoGenetics) relates to the nucleotidesequence encoding human Factor VII and the recombinant expression ofFactor VII in mammalian cells.

Dickinson et al. (Proc. Natl. Acad. Sci. USA (1996) 93, 14379-14384)relates to a Factor VII variant wherein Leu305 has been replaced by Ala(FVII(Ala305)).

Iwanaga et al. (Thromb. Haemost. (supplement August 1999), 466, abstract1474) relates to Factor VIIa variants wherein residues 316-320 aredeleted or residues 311-322 are replaced with the corresponding residuesfrom trypsin.

International patent applications WO 01/83725 and WO 02/22776 relates tovariants of Factor VIIa with increased activity.

There is a need for variants of Factor VIIa having coagulant activity,variants with high activity that can be administered at relatively lowdoses, and variants which do not produce the undesirable side effectssuch as systemic activation of the coagulation system and bleeding,respectively, associated with conventional therapies.

SUMMARY OF THE INVENTION

The present invention provides Factor VII polypeptides comprising atleast two substitutions relative to the amino acid sequence of SEQ IDNO:1, wherein said substitutions comprise: (i) a first substitutionconsisting of replacement of F374 with any other amino acid, and (ii)one or more substitutions consisting of replacement with any other aminoacid of one or more amino acids selected from the group consisting ofL305, S314, K157, K337, D334, S336, V158, E296, and M298. In someembodiments, both F374 and K157 are replaced with any other amino acid.In some embodiments, both F374 and K337 are replaced with any otheramino acid. In some embodiments, both F374 and D334 are replaced withany other amino acid. In some embodiments, both F374 and S336 arereplaced with any other amino acid. In some embodiments, both F374 andV158 are replaced with any other amino acid. In some embodiments, bothF374 and E296 are replaced with any other amino acid. In someembodiments, both F374 and M298 are replaced with any other amino. Insome embodiments, both F374 and L305 are replaced with any other aminoacid. In some embodiments, both F374 and S314 are replaced with anyother amino acid.

The invention also provides Factor VII polypeptides in which, inaddition to the above two substitutions, also comprise least oneadditional substitution at an amino acid residue in the protease domain,in which the additional residue has been replaced with any other aminoacid. In some embodiments, at most 20 additional amino acids in theremaining positions in the protease domain have been replaced with anyother amino acid. In different embodiments, the additional substitutionis at a residue corresponding to a position selected from 159-170;290-304; 306-312; or 330-339 of SEQ ID NO:1. In some embodiments, R304has been replaced by Tyr, Phe, Leu, or Met. In some embodiments, M306has been replaced by Asp or Asn. In some embodiments, D309 has beenreplaced by Ser or Thr. In some embodiments, wherein A274 has beenreplaced with any other amino acid, such as, e.g., Met, Leu, Lys, orArg.

The invention also encompasses Factor VII polypeptides with twosubstitutions relative to the amino acid sequence of SEQ ID NO:1,wherein said substitutions are (i) replacement of F374 with any otheramino acid and (ii) replacement with any other amino acid of one aminoacid selected from the group consisting of L305, S314, K157, K337, D334,S336, V158, E296, and M298. The invention also encompasses Factor VIIpolypeptides with three substitutions relative to the amino acidsequence of SEQ ID NO:1, wherein said substitutions are (i) replacementof F374 with any other amino acid and (ii) replacement with any otheramino acid of two amino acids selected from the group consisting ofL305, S314, K157, K337, D334, S336, V158, E296, and M298. The inventionalso encompasses Factor VII polypeptides with four substitutionsrelative to the amino acid sequence of SEQ ID NO:1, wherein saidsubstitutions are (i) replacement of F374 with any other amino acid and(ii) replacement with any other amino acid of three amino acids selectedfrom the group consisting of L305, S314, K157, K337, D334, S336, V158,E296, and M298. The invention also encompasses Factor VII polypeptideswith five substitutions relative to the amino acid sequence of SEQ IDNO:1, wherein said substitutions are (i) replacement of F374 with anyother amino acid and (ii) replacement with any other amino acid of fouramino acids selected from the group consisting of L305, S314, K157,K337, D334, S336, V158, E296, and M298. The invention also encompassesFactor VII polypeptides with six substitutions relative to the aminoacid sequence of SEQ ID NO:1, wherein said substitutions are (i)replacement of F374 with any other amino acid and (ii) replacement withany other amino acid of five amino acids selected from the groupconsisting of L305, S314, K157, K337, D334, S336, V158, E296, and M298.The invention also encompasses Factor VII polypeptides with sevensubstitutions relative to the amino acid sequence of SEQ ID NO: 1,wherein said substitutions are (i) replacement of F374 with any otheramino acid and (ii) replacement with any other amino acid of six aminoacids selected from the group consisting of L305, S314, K157, K337,D334, S336, V158, E296, and M298. The invention also encompasses FactorVII polypeptides with eight substitutions relative to the amino acidsequence of SEQ ID NO:1, wherein said substitutions are (i) replacementof F374 with any other amino acid and (ii) replacement with any otheramino acid of seven amino acids selected from the group consisting ofL305, S314, K157, K337, D334, S336, V158, E296, and M298. The inventionalso encompasses Factor VII polypeptides with nine substitutionsrelative to the amino acid sequence of SEQ ID NO:1, wherein saidsubstitutions are (i) replacement of F374 with any other amino acid and(ii) replacement with any other amino acid of eight amino acids selectedfrom the group consisting of L305, S314, K157, K337, D334, S336, V158,E296, and M298. The invention also encompasses Factor VII polypeptideswith ten substitutions relative to the amino acid sequence of SEQ IDNO:1, wherein said substitutions are (i) replacement of F374 with anyother amino acid and (ii) replacement with any other amino acid of theamino acids L305, S314, K157, K337, D334, S336, V158, E296, and M298.

In some embodiments of the invention, K157 has been replaced by Gly,Val, Ser, Thr, Asn, Gln, Asp, or Glu. In some embodiments, K337 has beenreplaced by Ala, Gly, Val, Ser, Thr, Asn, Gln, Asp, or Glu. In someembodiments, D334 has been replaced by Gly or Glu. In some embodiments,S336 has been replaced by Gly or Glu. In some embodiments, V158 has beenreplaced by Ser, Thr, Asn, Gln, Asp, or Glu. In some embodiments, E296has been replaced by Arg, Lys, Ile, Leu, or Val. In some embodiments,M298 has been re-placed by Lys, Arg, Gln, or Asn. In some embodiments,L305 has been replaced by Val, Tyr, or Ile. In some embodiments, S314has been replaced by Gly, Lys, Gln, or Glu.

In some embodiments, F374 has been replaced by Pro or Tyr.

In some embodiments, the Factor VII polypeptide is human Factor VII. Insome embodiments, the ratio between the activity of the Factor VIIpolypeptide of the invention and the activity of the native Factor VIIapolypeptide shown in SEQ ID NO:1 is at least about 1.25. In otherembodiments, this ratio is at least about 2.0, preferably at least about4.0.

In one embodiment, the Factor VII polypeptide F374Y/L305V-FVII. Inanother embodiment, the Factor VII polypeptide isF374Y/L305V/S314E/K337A-FVII. In another embodiment, the Factor VIIpolypeptide is F374Y/L305V/S314E-FVII. In another embodiment, the FactorVII polypeptide is F374Y/L305V/K337A-FVII.

The invention also encompasses polynucleotide constructs encoding theFactor VII polypeptides of the invention, vectors containing suchconstructs, and host cells comprising such constructs or vectors (whichmay be, but are not limited to, eukaryotic cells). In some embodiments,the host cells are mammalian cells, including, e.g., CHO cells, HEKcells and BHK cells.

The invention also encompasses transgenic animals and transgenic plantsthat express the Factor VII polypeptides of the invention.

The invention also encompasses methods for producing the Factor VIIpolypeptides of the invention, which are carried out by cultivating ahost cell as described above in an appropriate growth medium underconditions allowing expression of the polynucleotide construct andrecovering the resulting polypeptide from the culture medium. In anotherembodiment, the invention provides methods for producing the Factor VIIpolypeptides of the invention, which are carried out by recovering theFactor VII polypeptide from milk produced by a transgenic animal. Inanother embodiment, the invention provides methods for producing theFactor VII polypeptide of the invention, which are carried out bycultivating a cell of a transgenic plant and recovering the Factor VIIpolypeptide from the plant.

The present invention also encompasses pharmaceutical compositionscomprising a Factor VII polypeptide comprising at least twosubstitutions relative to the amino acid sequence of SEQ ID NO:1,wherein said substitutions are (i) replacement of F374 with any otheramino acid, and (ii) replacement with any other amino acid of one ormore amino acids selected from the group consisting of L305, S314, K157,K337, D334, S336, V158, E296, and M298; and, optionally, apharmaceutically acceptable carrier.

The present invention also encompasses the use of a Factor VIIpolypeptide comprising at least two substitutions relative to the aminoacid sequence of SEQ ID NO:1, wherein said substitutions are (i)replacement of F374 with any other amino acid, and (ii) replacement withany other amino acid of one or more amino acids selected from the groupconsisting of L305, S314, K157, K337, D334, S336, V158, E296, and M298;for one or more of: (a) the preparation of a medicament for thetreatment of bleeding disorders or bleeding episodes or for theenhancement of the normal haemostatic system; or (b) the treatment ofhaemophilia A or B.

The present invention also provides methods for the treatment ofbleeding disorders or bleeding episodes in a subject or for theenhancement of the normal haemostatic system, which are carried out byadministering a therapeutically or prophylactically effective amount ofa Factor VII polypeptide comprising at least two substitutions relativeto the amino acid sequence of SEQ ID NO:1, wherein said substitutionsare (i) replacement of F374 with any other amino acid, and (ii)replacement with any other amino acid of one or more amino acidsselected from the group consisting of L305, S314, K157, K337, D334,S336, V158, E296, and M298; to a subject in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the full amino acid sequence of native (wild type) humancoagulation Factor VII (SEQ ID NO:1).

FIG. 2 shows the sequence of oligonucleotide primers used for theconstruction of Factor VII variants.

DETAILED DESCRIPTION OF THE INVENTION

It has now been found that human coagulation Factor VIIa polypeptidevariants wherein the amino acid Phe374 and at least one amino acidindependently selected from the group consisting of Lys157, Leu305,Ser314, Lys337, Asp334, Ser336, Val 158, Glu296, and Met298 of SEQ IDNO:1 are replaced by different amino acids have increased coagulantactivity compared to wild type human coagulation Factor VIIa.

The term “a different amino acid” as used herein means one amino acidthat are different from that amino acid naturally present at thatposition. This includes, but is not limited to, amino acids that can beencoded by a polynucleotide. In one embodiment, the different amino acidis in natural L-form and can be encoded by a polynucleotide. A specificexample is L-cysteine (Cys).

The term “activity” as used herein means the ability of a Factor VIIpolypeptide to convert its substrate Factor X to the active Factor Xa.The activity of a Factor VII polypeptide may be measured with the “InVitro Proteolysis Assay” (see Example 4).

The term “inherent activity” also includes the ability to generatethrombin on the surface of activated platelets in the absence of tissuefactor.

Residue Phe374 is located at the end of an α-helix starting at residue307. This α-helix is found in the tissue factor-complexed form of FactorVIIA. In free Factor VIIa (Factor VIIa not bound to tissue factor), thehelix is distorted and thus possibly unstable. The helix is believed tobe important to the activity. Without wishing to be bound by theory, thevariants according to the present invention may attain the activeconformation, which normally has to be induced by tissue factor.

The increased activity may be due to a stabilisation of the α-helixstarting at residue 307, a reorientation of the helix or some otherchange in conformation. Replacement of the residue Phe374, which arelocated at the end of the helix, will induce a reorientation and/orstabilisation of the helix.

Due to the higher inherent activity of the described Factor VIla variantcompared to native Factor VIIa, a lower dose will be adequate to obtaina functionally adequate concentration at the site of action and thus itwill be possible to administer a lower dose to the subject havingbleeding episodes or needing enhancement of the normal haemostaticsystem.

Leu305 is located at the other end of this α-helix found in the tissuefactor-complexed form of Factor VIIa, which is believed to be importantto the activity. Replacement of the Leu305 may also induce areorientation and/or stabilisation of the helix.

The amino acids comprising Lys157, Ser314, Lys337, Asp334, Ser336, Val58, Glu296, and Met298 are located in an area believed to affect theinsertion of the amino terminus of the protease domain and thereby theformation of the catalytically active conformation of Factor VIIa whichis dependent on a salt bridge between the terminal amino group of Ile153and the side chain of Asp343. The replacements may remove electrostaticrepulsions, add hydrogen bonds or otherwise facilitate the insertion ofthe amino terminus.

Due to the higher inherent activity of the described Factor VIIapolypeptide variants compared to native VIIa, a lower dose may beadequate to obtain a functionally adequate concentration at the site ofaction and thus it will be possible to administer a lower dose to thesubject having bleeding episodes or needing enhancement of the normalhaemostatic system.

It has been found by the present inventors that by replacing the aminoacid Phe374 in combination with one or more of: Lys in position 157, Lysin position 337, Val in position 158, Glu in position 296, Met inposition 298, Asp in position 334, Ser in position 336, Leu in position305, and Ser in position 314, Factor VIIa will spontaneously attain amore active conformation that normally has to be induced by tissuefactor. Such Factor VIIa polypeptide variants exhibit an inherentactivity which may be therapeutically useful in situations where theprocoagulant activity is independent of tissue factor (Factor Xageneration on the platelet surface) such as when high doses of, forexample, NovoSeven® are administered.

In a further embodiment, additional replacement of amino acids in theprotease domain further facilitates formation of the active conformationof the molecule. It is believed, however, that the most pronouncedeffects will be seen when the above-mentioned mutations are carried outin the vicinity (sequential or three-dimensional) of these latter sevenamino acids.

The invention further comprises replacement of a few amino acids in theN-terminal Gla domain (amino acids at position corresponding to 1-37 ofSEQ ID NO:1) of Factor VIIa to provide the protein with a substantiallyhigher affinity for membrane phospholipids, such as membranephospholipids of tissue factor-bearing cells or of platelets, therebygenerating Factor VII polypeptide variants which have an improvedprocoagulant effect.

Thus, the Factor VIIa polypeptide variants mentioned above may, inaddition to the already performed amino acid replacement in positionF374 in combination with replacements in one or more of positions L305,S314, K157, K337, D334, S336, V158, E296, and M298 and the optionalamino acid replacements elsewhere in the protease domain, also have atleast one amino acid replaced in the N-terminal Gla domain, therebyobtaining a protein having an increased activity as well as an increasedaffinity for membrane phospholipids compared to native Factor VII.Preferably, the amino acids in positions 10 and 32 (referring to SEQ IDNO:1) of Factor VII may be replaced with a different amino acid.Examples of preferred amino acids to be incorporated in theabove-mentioned positions are: Pro in position 10 replaced by Gln, Arg,His, Gln, Asn or Lys; and/or Lys in position 32 replaced by Glu, Gln orAsn.

Other amino acids in the Gla domain, based on the different phospholipidaffinities and sequences of the vitamin K-dependent plasma proteins, mayalso be substituted.

The term “N-terminal GLA-domain” means the amino acid sequence 1-37 ofFactor The three-letter indication “GLA” means 4-carboxyglutamic acid(γcarboxyglutamate).

The term “protease domain” means the amino acid sequence 153-406 ofFactor VII (the heavy-chain of Factor VIIa).

The term “Factor VII polypeptide” as used herein means any proteincomprising the amino acid sequence 1-406 of native human Factor VII (SEQID NO: 1) or variants thereof. This includes, but is not limited to,human Factor VII, human Factor VIIa and variants thereof.

The term “Factor VII” as used herein is intended to comprise theinactive one-chain zymogen Factor VII molecule as well as the activatedtwo-chain Factor VII molecule (Factor VIIa). This includes proteins thathave the amino acid sequence 1-406 of native human Factor VII or FactorVIIa. It also includes proteins with a slightly modified amino acidsequence, for instance, a modified N-terminal end including N-terminalamino acid deletions or additions so long as those proteinssubstantially retain the activity of Factor VIIa. The term “factorVIIa”, or “FVIIa” as used herein means a product consisting of theactivated form (factor VIIa). “Factor VII” or “Factor VIIa” within theabove definition also includes natural allelic variations that may existand occur from one individual to another. Also, degree and location ofglycosylation or other post-translation modifications may vary dependingon the chosen host cells and the nature of the host cellularenvironment.

The terms “variant” or “variants”, as used herein, is intended todesignate Factor VII having the sequence of SEQ ID NO:1 wherein one ormore amino acids of the parent protein have been substituted by anotheramino acid and/or wherein one or more amino acids of the parent proteinhave been deleted and/or wherein one or more amino acids have beeninserted in protein and/or wherein one or more amino acids have beenadded to the parent protein. Such addition can take place either at theN-terminal end or at the C-terminal end of the parent protein or both.The “variant” or “variants” within this definition still have FVIIactivity in its activated form. In one embodiment, a variant is 70%identical with the sequence of of SEQ ID NO:1. In one embodiment, avariant is 80% identical with the sequence of of SEQ ID NO:1. In anotherembodiment, a variant is 90% identical with the sequence of of SEQ IDNO: 1. In a further embodiment, a variant is 95% identical with thesequence of of SEQ ID NO:1.

In a first aspect, the invention relates to a Factor VII polypeptidecomprising at least two substitutions relative to the amino acidsequence of SEQ ID NO:1, wherein said substitutions are (i) replacementof F374 with any other amino acid, and (ii) replacement with any otheramino acid of one or more amino acids selected from the group consistingof L305, S314, K157, K337, D334, S336, V158, E296, and M298.

In a second aspect, the invention relates to a Factor VII polypeptidewith two substitutions relative to the amino acid sequence of SEQ IDNO:1, wherein said substitutions are (i) replacement of F374 with anyother amino acid and (ii) replacement with any other amino acid of oneamino acid selected from the group consisting of L305, S314, K157, K337,D334, S336, V158, E296, and M298.

In a third aspect, the invention relates to a Factor VII polypeptidewith three substitutions relative to the amino acid sequence of SEQ IDNO: 1, wherein said substitutions are (i) replacement of F374 with anyother amino acid and (ii) replacement with any other amino acid of twoamino acids selected from the group consisting of L305, S314, K157,K337, D334, S336, V158, E296, and M298.

In a further aspect, the invention relates to a Factor VII polypeptidewith four substitutions relative to the amino acid sequence of SEQ IDNO:1, wherein said substitutions are (i) replacement of F374 with anyother amino acid and (ii) replacement with any other amino acid of threeamino acids selected from the group consisting of L305, S314, K157,K337, D334, S336, V158, E296, and M298.

In a further aspect, the invention relates to a Factor VII polypeptidewith five substitutions relative to the amino acid sequence of SEQ IDNO:1, wherein said substitutions are (i) replacement of F374 with anyother amino acid and (ii) replacement with any other amino acid of fouramino acids selected from the group consisting of L305, S314, K157,K337, D334, S336, V158, E296, and M298.

In a further aspect, the invention relates to a Factor VII polypeptidewith six substitutions relative to the amino acid sequence of SEQ IDNO:1, wherein said substitutions are (i) replacement of F374 with anyother amino acid and (ii) replacement with any other amino acid of fiveamino acids selected from the group consisting of L305, S314, K1157,K337, D334, S336, V158, E296, and M298.

In a further aspect, the invention relates to a Factor VII polypeptidewith seven substitutions relative to the amino acid sequence of SEQ IDNO: 1, wherein said substitutions are (i) replacement of F374 with anyother amino acid and (ii) replacement with any other amino acid of sixamino acids selected from the group consisting of L305, S314, K157,K337, D334, S336, V158, E296, and M298.

In a further aspect, the invention relates to a Factor VII polypeptidewith eight substitutions relative to the amino acid sequence of SEQ IDNO:1, wherein said substitutions are (i) replacement of F374 with anyother amino acid and (ii) replacement with any other amino acid of sevenamino acids selected from the group consisting of L305, S314, K157,K337, D334, S336, V158, E296, and M298.

In a further aspect, the invention relates to a Factor VII polypeptidewith nine substitutions relative to the amino acid sequence of SEQ IDNO:1, wherein said substitutions are (i) replacement of F374 with anyother amino acid and (ii) replacement with any other amino acid of eightamino acids selected from the group consisting of L305, S314, K157,K337, D334, S336, V158, E296, and M298.

In a further aspect, the invention relates to a Factor VII polypeptidewith ten substitutions relative to the amino acid sequence of SEQ IDNO:1, wherein said substitutions are (i) replacement of F374 with anyother amino acid and (ii) replacement with any other amino acid of theamino acids L305, S314, K157, K337, D334, S336, V158, E296, and M298.

In a further aspect, the invention relates to a polynucleotide constructencoding a Factor VII polypeptide comprising at least two substitutionsrelative to the amino acid sequence of SEQ ID NO:1, wherein saidsubstitutions are (i) replacement of F374 with any other amino acid, and(ii) replacement with any other amino acid of one or more amino acidsselected from the group consisting of L305, S314, K157, K337, D334,S336, V158, E296, and M298.

The term “construct” is intended to indicate a polynucleotide segmentwhich may be based on a complete or partial naturally occurringnucleotide sequence encoding the polypeptide of interest. The constructmay optionally contain other polynucleotide segments. In a similar way,the term “amino acids which can be encoded by polynucleotide constructs”covers amino acids which can be encoded by the polynucleotide constructsdefined above, i.e. amino acids such as Ala, Val, Leu, Ile, Met, Phe,Trp, Pro, Gly, Ser, Thr, Cys, Tyr, Asn, Glu, Lys, Arg, His, Asp and Gln.

In a further aspect, the invention provides a recombinant vectorcomprising the polynucleotide construct encoding a Factor VIIpolypeptide.

The term “vector”, as used herein, means any nucleic acid entity capableof the amplification in a host cell. Thus, the vector may be anautonomously replicating vector, i.e. a vector, which exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g. a plasmid. Alternatively, the vector maybe one which, when introduced into a host cell, is integrated into thehost cell genome and replicated together with the chromosome(s) intowhich it has been integrated. The choice of vector will often depend onthe host cell into which it is to be introduced. Vectors include, butare not limited to plasmid vectors, phage vectors, viruses or cosmidvectors. Vectors usually contains a replication origin and at least oneselectable gene, i.e., a gene which encodes a product which is readilydetectable or the presence of which is essential for cell growth.

In a further aspect, the invention provides a recombinant host cellcomprising the polynucleotide construct or the vector. In one embodimentthe recombinant host cell is a eukaryotic cell. In another embodimentthe recombinant host cell is of mammalian origin. In a furtherembodiment the recombinant host cell is selected from the groupconsisting of CHO cells, HEK cells and BHK cells.

The term “a host cell”, as used herein, represent any cell, includinghybrid cells, in which heterologous DNA can be expressed. Typical hostcells includes, but are not limited to insect cells, yeast cells,mammalian cells, including human cells, such as BHK, CHO, HEK, and COScells. In practicing the present invention, the host cells beingcultivated are preferably mammalian cells, more preferably anestablished mammalian cell line, including, without limitation, CHO(e.g., ATCC CCL 61), COS-1 (e.g., ATCC CRL 1650), baby hamster kidney(BHK) and HEK293 (e.g., ATCC CRL 1573; Graham et al., J. Gen. Virol.36:59-72, 1977) cell lines. A preferred BHK cell line is the tk⁻ ts13BHK cell line (Waechter and Baserga, Proc. Natl. Acad. Sci. USA79:1106-1110,1982), hereinafter referred to as BHK 570 cells. The BHK570 cell line is available from the American Type Culture Collection,12301 Parklawn Dr., Rockville, Md. 20852, under ATCC accession numberCRL 10314. A tk⁻ ts13 BHK cell line is also available from the ATCCunder accession number CRL 1632. Other suitable cell lines include,without limitation, Rat Hep I (Rat hepatoma; ATCC CRL 1600), Rat Hep II(Rat hepatoma; ATCC CRL 1548), TCMK (ATCC CCL 139), Human lung (ATCC HB8065), NCTC 1469 (ATCC CCL 9.1) and DUKX cells (Urlaub and Chasin, Proc.Natl. Acad. Sci. USA 77:4216-4220, 1980). Also useful are 3T3 cells,Namalwa cells, myelomas and fusions of myelomas with other cells.

In a further aspect, the invention provides a transgenic animalcontaining and expressing the polynucleotide construct.

In a further aspect, the invention provides a transgenic plantcontaining and expressing the polynucleotide construct.

In a further aspect, the invention relates to a method for producing theFactor VII polypeptide of the invention, the method comprisingcultivating a cell comprising the polynucleotide construct in anappropriate growth medium under conditions allowing expression of thepolynucleotide construct and recovering the resulting polypeptide fromthe culture medium.

As used herein the term “appropriate growth medium” means a mediumcontaining nutrients and other components required for the growth ofcells and the expression of the nucleic acid sequence encoding theFactor VII polypeptide of the invention.

In a further aspect, the invention relates to a method for producing theFactor VII polypeptide, the method comprising recovering the polypeptidefrom milk produced by the transgenic animal.

In a further aspect, the invention relates to a method for producing theFactor VIl polypeptide, the method comprising cultivating a cell of atransgenic plant comprising the polynucleotide construct, and recoveringthe polypeptide from the resulting plant.

In a further aspect, the invention relates to a pharmaceuticalcomposition comprising a Factor VII polypeptide comprising at least twosubstitutions relative to the amino acid sequence of SEQ ID NO:1,wherein said substitutions are (i) replacement of F374 with any otheramino acid, and (ii) replacement with any other amino acid of one ormore amino acids selected from the group consisting of L305, S314, K157,K337, D334, S336, V158, E296, and M298; and, optionally, apharmaceutically acceptable carrier.

In a further aspect, the invention relates to the use of a Factor VIIpolypeptide comprising at least two substitutions relative to the aminoacid sequence of SEQ ID NO:1, wherein said substitutions are (i)replacement of F374 with any other amino acid, and (ii) replacement withany other amino acid of one or more amino acids selected from the groupconsisting of L305, S314, K157, K337, D334, S336, V158, E296, and M298;for the preparation of a medicament for the treatment of bleedingdisorders or bleeding episodes or for the enhancement of the normalhaemostatic system. In one embodiment the use is for the treatment ofhaemophilia A or B.

In the present context, the term “treatment” is meant to include bothprevention of an expected bleeding, such as in surgery, and regulationof an already occurring bleeding, such as in trauma, with the purpose ofinhibiting or minimising the bleeding. Prophylactic administration ofthe Factor VIIa polypeptide according to the invention is thus includedin the term “treatment”.

The term “bleeding episodes” is meant to include uncontrolled andexcessive bleeding. Bleeding episodes may be a major problem both inconnection with surgery and other forms of tissue damage. Uncontrolledand excessive bleeding may occur in subjects having a normal coagulationsystem and subjects having coagulation or bleeding disorders. As usedherein the term “bleeding disorder” reflects any defect, congenital,acquired or induced, of cellular or molecular origin that is manifestedin bleedings. Examples are clotting factor deficiencies (e.g.haemophilia A and B or deficiency of coagulation Factors XI or VII),clotting factor inhibitors, defective platelet function,thrombocytopenia or von Willebrand's disease.

Excessive bleedings also occur in subjects with a normally functioningblood cloning cascade (no clotting factor deficiencies or -inhibitorsagainst any of the coagulation factors) and may be caused by a defectiveplatelet function, thrombocytopenia or von Willebrand's disease. In suchcases, the bleedings may be likened to those bleedings caused byhaemophilia because the haemostatic system, as in haemophilia, lacks orhas abnormal essential clotting “compounds” (such as platelets or vonWillebrand factor protein) that causes major bleedings. In subjects whoexperience extensive tissue damage in association with surgery or vasttrauma, the normal haemostatic mechanism may be overwhelmed by thedemand of immediate haemostasis and they may develop bleeding in spiteof a normal haemostatic mechanism. Achieving satisfactory haemostasisalso is a problem when bleedings occur in organs such as the brain,inner ear region and eyes with limited possibility for surgicalhaemostasis. The same problem may arise in the process of takingbiopsies from various organs (liver, lung, tumour tissue,gastrointestinal tract) as well as in laparoscopic surgery. Common forall these situations is the difficulty to provide haemostasis bysurgical techniques (sutures, clips, etc.) which also is the case whenbleeding is diffuse (haemorrhagic gastritis and profuse uterinebleeding). Acute and profuse bleedings may also occur in subjects onanticoagulant therapy in whom a defective haemostasis has been inducedby the therapy given. Such subjects may need surgical interventions incase the anticoagulant effect has to be counteracted rapidly. Radicalretropubic prostatectomy is a commonly performed procedure for subjectswith localized prostate cancer. The operation is frequently complicatedby significant and sometimes massive blood loss. The considerable bloodloss during prostatectomy is mainly related to the complicatedanatomical situation, with various densely vascularized sites that arenot easily accessible for surgical haemostasis, and which may result indiffuse bleeding from a large area. Another situation that may causeproblems in the case of unsatisfactory haemostasis is when subjects witha normal haemostatic mechanism are given anticoagulant therapy toprevent thromboembolic disease. Such therapy may include heparin, otherforms of proteoglycans, warlarin or other forms of vitamin K-antagonistsas well as aspirin and other platelet aggregation inhibitors.

In one embodiment of the invention, the bleeding is associated withhaemophilia. In another embodiment, the bleeding is associated withhaemophilia with aquired inhibitors. In another embodiment, the bleedingis associated with thrombocytopenia. In another embodiment, the bleedingis associated with von Willebrand's disease. In another embodiment, thebleeding is associated with severe tissue damage. In another embodiment,the bleeding is associated with severe trauma. In another embodiment,the bleeding is associated with surgery. In another embodiment, thebleeding is associated with laparoscopic surgery. In another embodiment,the bleeding is associated with haemorrhagic gastritis. In anotherembodiment, the bleeding is profuse uterine bleeding. In anotherembodiment, the bleeding is occurring in organs with a limitedpossibility for mechanical haemostasis. In another embodiment, thebleeding is occurring in the brain, inner ear region or eyes. In anotherembodiment, the bleeding is associated with the process of takingbiopsies. In another embodiment, the bleeding is associated withanticoagulant therapy.

The term “subject” as used herein is intended to mean any animal, inparticular mammals, such as humans, and may, where appropriate, be usedinterchangeably with the term “patient”.

The term “enhancement of the normal haemostatic system” means anenhancement of the ability to generate thrombin.

In a further aspect, the invention relates to a method for the treatmentof bleeding disorders or bleeding episodes in a subject or for theenhancement of the normal haemostatic system, the method comprisingadministering a therapeutically or prophylactically effective amount ofa Factor VII polypeptide comprising at least two substitutions relativeto the amino acid sequence of SEQ ID NO:1, wherein said substitutionsare (i) replacement of F374 with any other amino acid, and (ii)replacement with any other amino acid of one or more amino acidsselected from the group consisting of L305, S314, K157, K337, D334,S336, V158, E296, and M298; to a subject in need thereof.

In a further aspect, the invention relates to the Factor VII polypeptideof the invention for use as a medicament.

In one embodiment of the invention, the factor VII polypeptide is apolypeptide, wherein F374 is replaced with any other amino acid and L305is replaced with any other amino acid.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein F374 is replaced with any other amino acid andS314 is replaced with any other amino acid.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein F374 is replaced with any other amino acid andK157 is replaced with any other amino acid.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein F374 is replaced with any other amino acid andK337 is replaced with any other amino acid.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein F374 is replaced with any other amino acid andD334 is replaced with any other amino acid.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein F374 is replaced with any other amino acid andS336 is replaced with any other amino acid.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein F374 is replaced with any other amino acid andV158 is replaced with any other amino acid.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein F374 is replaced with any other amino acid andE296 is replaced with any other amino acid.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein F374 is replaced with any other amino acid andM298 is replaced with any other amino acid.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein at least one amino acid in the remainingpositions in the protease domain has been replaced with any other aminoacid. In one embodiment, the factor VII polypeptide is a polypeptide,wherein one amino acid in the remaining positions in the protease domainhas been replaced with any other amino acid.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein at the most 20 additional amino acids in theremaining positions in the protease domain have been replaced with anyother amino acids.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein at least one amino acid corresponding to an aminoacid at a position selected from 159-170 of SEQ ID NO:1 has beenreplaced with any other amino acid.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein at least one amino acid corresponding to an aminoacid at a position selected from 290-304 of SEQ ID NO:1 has beenreplaced with any other amino acid.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein R304 has been replaced with any other amino acid.In one embodiment R304 has been replaced by an amino acid selected fromthe group consisting of Tyr, Phe, Leu, and Met.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein at least one amino acid corresponding to an aminoacid at a position selected from 306-312 of SEQ ID NO:1 has beenreplaced with any other amino acid.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein M306 has been replaced by an amino acid selectedfrom the group consisting of Asp, and Asn.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein D309 has been replaced by an amino acid selectedfrom the group consisting of Ser, and Thr.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein at least one amino acid corresponding to an aminoacid at a position selected from 330-339 of SEQ ID NO:1 has beenreplaced with any other amino acid.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein A274 has been replaced with any other amino acid.

In a further embodiment of the invention, the factor VlI polypeptide isa polypeptide, wherein the A274 has been replaced by an amino acidselected from the group consisting of Met, Leu, Lys, and Arg.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein the K157 has been replaced by an amino acidselected from the group consisting of Ala, Val, Leu, Ile, Met, Phe, Trp,Pro, Gly, Ser, Thr, Cys, Tyr, Asn, Glu, Arg, His, Asp and Gln.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein the K157 has been replaced by an amino acidselected from the group consisting of Gly, Val, Ser, Thr, Asn, Gln, Asp,and Glu.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein said K337 has been replaced by an amino acidselected from the group consisting of Ala, Val, Leu, Ile, Met, Phe, Trp,Pro, Gly, Ser, Thr, Cys, Tyr, Asn, Glu, Arg, His, Asp and Gln.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein said K337 has been replaced by an amino acidselected from the group consisting of Ala, Gly, Val, Ser, Thr, Asn, Gln,Asp, and Glu.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein said D334 has been replaced by an amino acidselected from the group consisting of Ala, Val, Leu, Ile, Met, Phe, Trp,Pro, Gly, Ser, Thr, Cys, Tyr, Asn, Glu, Lys, Arg, His, and Gln.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein said D334 has been replaced by an amino acidselected from the group consisting of Gly, and Glu.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein said S336 has been replaced by an amino acidselected from the group consisting of Ala, Val, Leu, Ile, Met, Phe, Trp,Pro, Gly, Thr, Cys, Tyr, Asn, Glu, Lys, Arg, His, Asp and Gln.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein said S336 has been replaced by an amino acidselected from the group consisting of Gly, and Glu.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein said V158 has been replaced by an amino acidselected from the group consisting of Ala, Leu, Ile, Met, Phe, Trp, Pro,Gly, Ser, Thr, Cys, Tyr, Asn, Glu, Lys, Arg, His, Asp and Gln.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein said V158 has been replaced by an amino acidselected from the group consisting of Ser, Thr, Asn, Gln, Asp, and Glu.In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein said E296 has been replaced by an amino acidselected from the group consisting of Ala, Val, Leu, Ile, Met, Phe, Trp,Pro, Gly, Ser, Thr, Cys, Tyr, Asn, Lys, Arg, His, Asp and Gln.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein said E296 has been replaced by an amino acidselected from the group consisting of Arg, Lys, and Val.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein said M298 has been replaced by an amino acidselected from the group consisting of Ala, Val, Leu, Ile, Phe, Trp, Pro,Gly, Ser, Thr, Cys, Tyr, Asn, Glu, Lys, Arg, His, Asp and Gln.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein said M298 has been replaced by an amino acidselected from the group consisting of Lys, Arg, Gln, and Asn.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein said L305 has been replaced by an amino acidselected from the group consisting of Ala, Val, Ile, Met, Phe, Trp, Pro,Gly, Ser, Thr, Cys, Tyr, Asn, Glu, Lys, Arg, His, Asp and Gln.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein said L305 has been replaced by an amino acidselected from the group consisting of Val, Tyr and Ile.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein said L305 has been replaced by Val.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein said S314 has been replaced by an amino acidselected from the group consisting of Ala, Val, Leu, Ile, Met, Phe, Trp,Pro, Gly, Thr, Cys, Tyr, Asn, Glu, Lys, Arg, His, Asp and Gln.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein said S314 has been replaced by an amino acidselected from the group consisting of Gly, Lys, Gln and Glu.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein said F374 has been replaced by an amino acidselected from the group consisting of Ala, Val, Leu, Ile, Met, Trp, Pro,Gly, Ser, Thr, Cys, Tyr, Asn, Glu, Lys, Arg, His, Asp and Gln.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein said F374 has been replaced by an amino acidselected from the group consisting of Pro and Tyr.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein the amino acid has been replaced by a differentamino acid which can be encoded by polynucleotide constructs.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein said Factor VII polypeptide is human Factor VII.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein said Factor VII polypeptide is human Factor VIIa.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein the ratio between the activity of said Factor VIIpolypeptide and the activity of the native Factor VIIa polypeptide shownin SEQ ID NO:1 is at least about 1.25. In one embodiment the ratiobetween the activity of said Factor VII polypeptide and the activity ofthe native Factor VIIa polypeptide shown in SEQ ID NO:1 is at leastabout 2.0. In a further embodiment the ratio between the activity ofsaid Factor VII polypeptide and the activity of the native Factor VIIapolypeptide shown in SEQ ID NO:1 is at least about 4.0.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein the ratio between the activity of said Factor VIIpolypeptide and the activity of the native Factor VIIa polypeptide shownin SEQ ID NO:1 is at least about 1.25 when tested in a Factor VIIaactivity assay. In one embodiment the ratio between the activity of saidFactor VII polypeptide and the activity of the native Factor VIIapolypeptide shown in SEQ ID NO:1 is at least about 2.0 when tested in aFactor VIIa activity assay. In a further embodiment the ratio betweenthe activity of said Factor VII polypeptide and the activity of thenative Factor VIIa polypeptide shown in SEQ ID NO:1 is at least about4.0 when tested in a Factor VIIa activity assay. The Factor VIIaactivity may be measured by the assays described in examples 3 or 4.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein the ratio between the activity of said Factor VIIpolypeptide and the activity of the native Factor VIIa polypeptide shownin SEQ ID NO:1 is at least about 1.25 when tested in the “In VitroHydrolysis Assay”. In one embodiment the ratio between the activity ofsaid Factor VII polypeptide and the activity of the native Factor VIIapolypeptide shown in SEQ ID NO:1 is at least about 2.0 when tested inthe “In Vitro Hydrolysis Assay”. In a further embodiment the ratiobetween the activity of said Factor VII polypeptide and the activity ofthe native Factor VIIa polypeptide shown in SEQ ID NO:1 is at leastabout 4.0 when tested in the “In Vitro Hydrolysis Assay”.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein the ratio between the activity of said Factor VIIpolypeptide and the activity of the native Factor VIIa polypeptide shownin SEQ ID NO: 1 is at least about 1.25 when tested in the “In VitroProteolysis Assay”. In one embodiment the ratio between the activity ofsaid Factor VII polypeptide and the activity of the native Factor VIIapolypeptide shown in SEQ ID NO:1 is at least about 2.0 when tested inthe “In Vitro Proteolysis Assay”. In a further embodiment the ratiobetween the activity of said Factor VII polypeptide and the activity ofthe native Factor VII polypeptide shown in SEQ ID NO:1 is at least about4.0 when tested in the “In Vitro Proteolysis Assay”. In a furtherembodiment the ratio between the activity of said Factor VII polypeptideand the activity of the native Factor VIIa polypeptide shown in SEQ IDNO:1 is at least about 8.0 when tested in the “In Vitro ProteolysisAssay”.

In a further embodiment of the invention, the factor VII polypeptide ishuman FVII with at least two substitutions relative to the amino acidsequence of SEQ ID NO:1, wherein said substitutions are (i) F374Y and(ii) one or more amino acids selected from the group consisting ofK157X¹, K337A, D334X², S336X³, V158X⁴, E296V, M298Q, L305V, S314E,wherein X¹ is Gly, Val, Ser, Thr, Asn, Gln, Asp, or Glu; X² is Gly orGlu; X³ is Gly or Glu; X⁴ is Thr or Asp.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/K337A-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/V158D-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/E296V-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/M298Q-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/M158T-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/K337A-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/V158D-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/E296V-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/M298Q-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/V158T-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/K337A/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/K337A/V158T-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/K337A/M298Q-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/K337A/E296V-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/K337A/V158D-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/V158D/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/V158D/M298Q-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/V158D/E296V-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/V158T/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/V 158T/M298Q-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/V 158T/E296V-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/E296V/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/S314E/M298Q-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/E296V/M298Q-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/K337A/V158D-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/K337A/E296V-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/K337A/M298Q-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/K337A/V158T-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/K337A/S314E-FVII.

In a further embodiment of the Invention, the factor VII polypeptide isF374Y/L305V/V158D/E296V-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/V158D/M298Q-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/V158D/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/E296V/M298Q-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/E296V/V158T-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/E296V/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/M298Q/V158T-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/M298Q/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/V158T/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/K337A/S314E/V158T-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/K337A/S314E/M298Q-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/K337A/S314E/E296V-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/K337A/S314E/V158D-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/K337A/V158T/M298Q-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/K337A/V158T/E296V-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/K337A/M298Q/E296V-FVII.

In a further embodiment of the invention, the factor V polypeptide isF374Y/K337A/M298Q/V158D-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/K337A/E296V/V158D-FVII.

In a further embodiment of the invention, the factor VII polypeptide is,F374Y/V158D/S314E/M298Q-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/V158D/S314E/E296V-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/V158D/M298Q/E296V-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/V158T/S314E/E296V-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/V158T/S314E/M298Q-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/V158T/M298Q/E296V-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/E296V/S314E/M298Q-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/M298Q/K337A/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/E296V/K337A/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide is,F374Y/E296V/M298Q/K337A/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/E296V/M298Q/K337A-VII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/E296V/M298Q/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/V158D/E296V/M298Q/K337A-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/V158D/E296V/M298Q/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/V158D/K337A/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/V158D/M298Q/K337A/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374V/V158D/E296V/K337A/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/V158D/E296V/M298Q-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/V158D/M298Q/K337A-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/V158D/E296V/K337A-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/V158D/M298Q/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/V158D/E296V/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/V158T/E296V/M298Q/K337A-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/V158T/E296V/M298Q/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/V158T/K337A/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/V158T/M298Q/K337A/S314E-FVII.

In a further embodiment of the invention, the factor VlI polypeptide isF374Y/V158T/E296V/K337A/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/V158T/E296V/M298Q-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/V158T/M298Q/K337A-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/V158T/E296V/K337A-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/V158T/M298Q/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/V158T/E296V/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/E296V/M298Q/K337A/V158T/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/V158D/E296V/M298Q/K337A/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/V158D/E296V/M298Q/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/E296V/M298Q/V158T/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/E296V/M298Q/K337A/V158T-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/E296V/K337A/V158T/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/M298Q/K337A/V158T/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/V158D/E296V/M298Q/K337A-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/V158D/E296V/K337A/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/V158D/M298Q/K337A/S314E-FVII.

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/E296V/M298Q/K337A/V158T/S314E-FVII

In a further embodiment of the invention, the factor VII polypeptide isF374Y/L305V/V158D/E296V/M298Q/K337A/S314E-FVII

In a further embodiment of the invention, the factor VII polypeptide ishuman FVII with (i) the amino acid substitution F374Y relative to theamino acid sequence of SEQ ID NO:1, and (ii) two amino acidsubstitutions independently selected from any combination according totable 1.

In a further embodiment of the invention, the factor VII polypeptide ishuman FVII with (i) the amino acid substitution F374Y relative to theamino acid sequence of SEQ ID NO:1, and (ii) three amino acidsubstitutions independently selected from any combination according totable 1.

In a further embodiment of the invention, the factor VII polypeptide ishuman FVII with (i) the amino acid substitution F374Y relative to theamino acid sequence of SEQ ID NO:1, and (ii) four amino acidsubstitutions independently selected from any combination according totable 1.

In a further embodiment of the invention, the factor VI) polypeptide ishuman FVII with (i) the amino acid substitution F374Y relative to theamino acid sequence of SEQ ID NO:1, and (ii) five amino acidsubstitutions independently selected from any combination according totable 1.

In a further embodiment of the invention, the factor VII polypeptide ishuman FVII with (i) the amino acid substitution F374Y relative to theamino acid sequence of SEQ ID NO:1, and (ii) six amino acidsubstitutions independently selected from any combination according totable 1.

In a further embodiment of the invention, the factor VII polypeptide ishuman FVII with (i) the amino acid substitution F374Y relative to theamino acid sequence of SEQ ID NO:1, and (ii) seven amino acidsubstitutions independently selected from any combination according totable 1.

In a further embodiment of the invention, the factor VII polypeptide ishuman FVII with (i) the amino acid substitution F374Y relative to theamino acid sequence of SEQ ID NO: 1, and (ii) eight amino acidsubstitutions independently selected from any combination according totable 1.

In a further embodiment of the invention, the factor VII polypeptide ishuman FVII with (i) the amino acid substitution F374Y relative to theamino acid sequence of SEQ ID acid substitutions independently selectedfrom any combination according to table 1.

TABLE 1 Amino acid substitution relative to the amino acid sequence ofSEQ ID NO:1. L305V S314E K337A V158D V158T E296V M298Q S336E S336G D334EL305V n + + + + + + + + + S314E + n + + + + + + + + K337A + +n + + + + + + + V158D + + + n n + + + + + V158T + + + n n + + + + +E296V + + + + + n + + + + M298Q + + + + + + n + + + S336E + + + + + + +n n + S336G + + + + + + + n n + D334E + + + + + + + + + nD334G + + + + + + + + + n K157E + + + + + + + + + +K157D + + + + + + + + + + K157Q + + + + + + + + + +K157N + + + + + + + + + + K157T + + + + + + + + + +K157S + + + + + + + + + + K157V + + + + + + + + + +K157G + + + + + + + + + + Amino acid substitution relative to the aminoacid sequence of SEQ ID NO:1. D334G K157E K157D K157Q K157N K157T K157SK157V K157G L305V + + + + + + + + + S314E + + + + + + + + +K337A + + + + + + + + + V158D + + + + + + + + + V158T + + + + + + + + +E296V + + + + + + + + + M298Q + + + + + + + + + S336E + + + + + + + + +S336G + + + + + + + + + D334E n + + + + + + + + D334G n + + + + + + + +K157E + n n n n n n n n K157D + n n n n n n n n K157Q + n n n n n n n nK157N + n n n n n n n n K157T + n n n n n n n n K157S + n n n n n n n nK157V + n n n n n n n n K157G + n n n n n n n n (+: valid combination,n: Not applicable)

In a further aspect, the invention provides human coagulation FactorVIIa polypeptides that have increased tissue factor-independent activitycompared to native human coagulation Factor VIIa. In another aspect, theincreased activity is not accompanied by changes in the substratespecificity. In another aspect of the invention, the binding of thepolypeptide variants to tissue factor should not be impaired and thepolypeptide variants should have at least the activity of wild-typeFactor VIA when bound to tissue factor.

The terminology for amino acid substitutions used in this descriptionare as follows. The first letter represent the amino acid naturallypresent at a position of SEQ ID NO:1. The following number represent theposition in SEQ ID NO:1. The second letter represent the different aminoacid substituting for the natural amino acid. An example isL305V/K337A-FVII, the leucine at position 305 of SEQ ID NO:1 is replacedby a valine and the Lysine at position 337 of SEQ ID NO:1 is replaced byan alanine, both mutations in the same Factor VII polypeptide variant.

In the present context the three-letter or one-letter indications of theamino acids have been used in their conventional meaning as indicated intable 2. Unless indicated explicitly, the amino acids mentioned hereinare L-amino acids. Further, the left and right ends of an amino acidsequence of a peptide are, respectively, the N- and C-tenmini unlessotherwise speci fied.

TABLE 2 Abbreviations for amino acids: Amino acid Tree-letter codeOne-letter code Glycine Gly G Proline Pro P Alanine Ala A Valine Val VLeucine Leu L Isoleucine Ile I Methionine Met M Cysteine Cys CPhenylalanine Phe F Tyrosine Tyr Y Tryptophan Trp W Histidine His HLysine Lys K Arginine Arg R Glutamine Gln Q Asparagine Asn N GlutamicAcid Glu E Aspartic Acid Asp D Serine Ser S Threonine Thr TPreparation of Factor VII polyeptide variants The invention also relatesto a method of preparing human Factor VII polypeptide variants asmentioned above. The Factor VII polypeptide variants described hereinmay be produced by means of recombinant nucleic acid techniques. Ingeneral, a cloned wild-type Factor VII nucleic acid sequence is modifiedto encode the desired protein. This modified sequence is then insertedinto an expression vector, which is in turn transformed or transfectedinto host cells. Higher eukaryotic cells, in particular culturedmammalian cells, are preferred as host cells. The complete nucleotideand amino acid sequences for human Factor VII are known (see U.S. Pat.No. 4,784,950, where the cloning and expression of recombinant humanFactor VII is described). The bovine Factor VII sequence is described inTakeya et al., J. Biol. Chem. 263:14868-14872 (1988)).

The amino acid sequence alterations may be accomplished by a variety oftechniques. Modification of the nucleic acid sequence may be bysite-specific mutagenesis. Techniques for site-specific mutagenesis arewell known in the art and are described in, for example, Zoller andSmith (DNA 3:479-488, 1984) or “Splicing by extension overlap”, Hortonet al., Gene 77,1989, pp. 61-68. Thus, using the nucleotide and aminoacid sequences of Factor VII, one may introduce the alteration(s) ofchoice. Likewise, procedures for preparing a DNA construct usingpolymerase chain reaction using specific primers are well known topersons skilled in the art (cf. PCR Protocols, 1990, Academic Press, SanDiego, Calif., USA).

The nucleic acid construct encoding the Factor VII polypeptide variantof the invention may suitably be of genomic or cDNA origin, for instanceobtained by preparing a genomic or cDNA library and screening for DNAsequences coding for all or part of the polypeptide by hybridizationusing synthetic oligonucleotide probes in accordance with standardtechniques (cf. Sambrook et al., Molecular Cloning: A Laboratory Manual,2nd. Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).

The nucleic acid construct encoding the Factor VII polypeptide variantmay also be prepared synthetically by established standard methods, e.g.the phosphoamidite method described by Beaucage and Caruthers,Tetrahedron Letters 22 (1981), 1859-1869, or the method described byMatthes et al., EMBO Journal 3 (1984), 801-805. According to thephosphoamidite method, oligonucleotides are synthesised, e.g. in anautomatic DNA synthesiser, purified, annealed, ligated and cloned insuitable vectors. The DNA sequences encoding the human Factor VIIpolypeptide variants may also be prepared by polymerase chain reactionusing specific primers, for instance as described in U.S. Pat. No.4,683,202, Saiki et al., Science 239 (1988), 487-491, or Sambrook etal., supra.

Furthermore, the nucleic acid construct may be of mixed synthetic andgenomic, mixed synthetic and cDNA or mixed genomic and cDNA originprepared by ligating fragments of synthetic, genomic or cDNA origin (asappropriate), the fragments corresponding to various parts of the entirenucleic acid construct, in accordance with standard techniques.

The nucleic acid construct is preferably a DNA construct. DNA sequencesfor use in producing Factor VII polypeptide variants according to thepresent invention will typically encode a pre-pro polypeptide at theamino-terminus of Factor VII to obtain proper posttranslationalprocessing (e.g. gamma-carboxylation of glutamic acid residues) andsecretion from the host cell. The pre-pro polypeptide may be that ofFactor VII or another vitamin K-dependent plasma protein, such as FactorIX, Factor X, prothrombin, protein C or protein S. As will beappreciated by those skilled in the art, additional modifications can bemade in the amino acid sequence of the Factor VII polypeptide variantswhere those modifications do not significantly impair the ability of theprotein to act as a coagulant. For example, the Factor VII polypeptidevariants can also be modified in the activation cleavage site to inhibitthe conversion of zymogen Factor VII into its activated two-chain form,as generally described in U.S. Pat. No. 5,288,629, incorporated hereinby reference.

The DNA sequences encoding the human Factor VII polypeptide variants areusually inserted into a recombinant vector which may be any vector,which may conveniently be subjected to recombinant DNA procedures, andthe choice of vector will often depend on the host cell into which it isto be introduced. Thus, the vector may be an autonomously replicatingvector, i.e. a vector, which exists as an extrachromosomal entity, thereplication of which is independent of chromosomal replication, e.g. aplasmid. Alternatively, the vector may be one which, when introducedinto a host cell, is integrated into the host cell genome and replicatedtogether with the chromosome(s) into which it has been integrated.

The vector is preferably an expression vector in which the DNA sequenceencoding the human Factor VII polypeptide variants is operably linked toadditional segments required for transcription of the DNA. In general,the expression vector is derived from plasmid or viral DNA, or maycontain elements of both. The term, “operably linked” indicates that thesegments are arranged so that they function in concert for theirintended purposes, e.g. transcription initiates in a promoter andproceeds through the DNA sequence coding for the polypeptide.

Expression vectors for use in expressing Factor VII polypeptide variantswill comprise a promoter capable of directing the transcription of acloned gene or cDNA. The promoter may be any DNA sequence, which showstranscriptional activity in the host cell of choice and may be derivedfrom genes encoding proteins either homologous or heterologous to thehost cell.

Examples of suitable promoters for directing the transcription of theDNA encoding the human Factor VII polypeptide variant in mammalian cellsare the SV40 promoter (Subramani et al., Mol. Cell Biol. 1 (1981),854-864), the MT-1 (metallothionein gene) promoter (Palmiter et al.,Science 222 (1983), 809-814), the CMV promoter (Boshart et al., Cell41:521-530, 1985) or the adenovirus 2 major late promoter (Kaufman andSharp, Mol. Cell. Biol, 2:1304-1319, 1982).

An example of a suitable promoter for use in insect cells is thepolyhedrin promoter (U.S. Pat. No. 4,745,051; Vasuvedan et al., FEBSLett. 311, (1992) 7-11), the P10 promoter (J. M. Vlak et al., J. Gen.Virology 69, 1988, pp. 765-776), the Autographa californica polyhedrosisvirus basic protein promoter (EP 397 485), the baculovirus immediateearly gene 1 promoter (U.S. Pat. No. 5,155,037; U.S. Pat. No.5,162,222), or the baculovirus 39K delayed-early gene promoter (U.S.Pat. No. 5,155,037; U.S. Pat. No. 5,162,222).

Examples of suitable promoters for use in yeast host cells includepromoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem.255 (1980), 12073-12080; Alber and Kawasaki, J. Mol. Appl. Gen. 1(1982), 419-434) or alcohol dehydrogenase genes (Young et al., inGenetic Engineering of Microorganisms for Chemicals (Hollaender et al,eds.), Plenum Press, New York, 1982), or the TPI1 (U.S. Pat. No.4,599,311) or ADH2-4c (Russell et al., Nature 304 (1983), 652-654)promoters.

Examples of suitable promoters for use in filamentous fungus host cellsare, for instance, the ADH3 promoter (McKnight et al., The EMBO J. 4(1985), 2093-2099) or the tpiA promoter. Examples of other usefulpromoters are those derived from the gene encoding A olyzae TAKAamylase, Rhizomucor miehei aspartic proteinase, A. niger neutralα-amylase, A. niger acid stable o-amylase, A. niger or A. awamoriglucoamylase (gluA), Rhizomucor miehei lipase, A. oryzae alkalineprotease, A. oryzae triose phosphate isomerase or A. nidulansacetamidase. Preferred are the TAKA-amylase and gluA promoters. Suitablepromoters are mentioned In, e.g. EP 238 023 and EP 383 779.

The DNA sequences encoding the human Factor Vi polypeptide variants mayalso, if necessary, be operably connected to a suitable terminator, suchas the human growth hormone terminator (Palmiter et al., Science 222,1983, pp. 809-814) or the TPI 1 (Alber and Kawasaki, J. Mol. Appl. Gen.1,1982, pp. 419-434) or ADH3 (McKnight et al., The EMBO J. 4,1985, pp.2093-2099) terminators. Expression vectors may also contain a set of RNAsplice sites located downstream from the promoter and upstream from theinsertion site for the Factor VII sequence itself. Preferred RNA splicesites may be obtained from adenovirus and/or immunoglobulin genes. Alsocontained in the expression vectors is a polyadenylation signal locateddownstream of the insertion site. Particularly preferred polyadenylationsignals include the early or late polyadenylation signal from SV40(Kaufman and Sharp, ibid.), the polyadenylation signal from theadenovirus 5 E1b region, the human growth hormone gene terminator(DeNoto et al. Nucl. Acids Res. 9:37193730, 1981) or the polyadenylationsignal from the human Factor VII gene or the bovine Factor VII gene. Theexpression vectors may also include a noncoding viral leader sequence,such as the adenovirus 2 tripartite leader, located between the promoterand the RNA splice sites; and enhancer sequences, such as the SV40enhancer.

To direct the human Factor VII polypeptide variants of the presentinvention into the secretory pathway of the host cells, a secretorysignal sequence (also known as a leader sequence, prepro sequence or presequence) may be provided in the recombinant vector. The secretorysignal sequence is joined to the DNA sequences encoding the human FactorVII polypeptide variants in the correct reading frame. Secretory signalsequences are commonly positioned 5′ to the DNA sequence encoding thepeptide. The secretory signal sequence may be that, normally associatedwith the protein or may be from a gene encoding another secretedprotein.

For secretion from yeast cells, the secretory signal sequence may encodeany signal peptide, which ensures efficient direction of the expressedhuman Factor VII polypeptide variants into the secretory pathway of thecell. The signal peptide may be naturally occurring signal peptide, or afunctional part thereof, or it may be a synthetic peptide. Suitablesignal peptides have been found to be the a factor signal peptide (cf.U.S. Pat. No. 4,870,008), the signal peptide of mouse salivary amylase(cf. 0. Hagenbuchle et al., Nature 289,1981, pp. 643646), a modifiedcarboxypeptidase signal peptide (cf. L. A. Valls et al., Cell 48, 1987,pp. 887-897), the yeast BAR1 signal peptide (cf. WO 87/02670), or theyeast aspartic protease 3 (YAP3) signal peptide (cf. M. Egel-Mitani etal., Yeast 6, 1990, pp. 127-137).

For efficient secretion in yeast, a sequence encoding a leader peptidemay also be inserted downstream of the signal sequence and upstream ofthe DNA sequence encoding the human Factor VII polypeptide variants. Thefunction of the leader peptide is to allow the expressed peptide to bedirected from the endoplasmic reticulum to the Golgi apparatus andfurther to a secretory vesicle for secretion into the culture medium(i.e. exportation of the human Factor VII polypeptide variants acrossthe cell wall or at least through the cellular membrane into theperiplasmic space of the yeast cell). The leader peptide may be theyeast alpha-factor leader (the use of which is described in e.g. U.S.Pat. No. 4,546,082, U.S. Pat. No. 4,870,008, EP 16 201, EP 123 294, EP123 544 and EP 163 529). Alternatively, the leader peptide may be asynthetic leader peptide, which is to say a leader peptide not found innature. Synthetic leader peptides may, for instance, be constructed asdescribed in WO 89/02463 or WO 92/11378.

For use in filamentous fungi, the signal peptide may conveniently bederived from a gene encoding an Aspergillus sp. amylase or glucoamylase,a gene encoding a Rhizomucor miehei lipase or protease or a Humicolalanuginosa lipase. The signal peptide is preferably derived from a geneencoding A. oryzae TAKA amylase, A. niger neutral α-amylase, A. nigeracid-stable amylase, or A. niger glucoamylase. Suitable signal peptidesare disclosed in, e.g. EP 238 023 and EP 215 594.

For use in insect cells, the signal peptide may conveniently be derivedfrom an insect gene (cf. WO 90/05783), such as the lepidopteran Manducasexta adipokinetic hormone precursor signal peptide (cf. U.S. Pat. No.5,023,328).

The procedures used to ligate the DNA sequences coding for the humanFactor VII polypeptide variants, the promoter and optionally theterminator and/or secretory signal sequence, respectively, and to insertthem into suitable vectors containing the information necessary forreplication, are well known to persons skilled in the art (cf., forinstance, Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor, N.Y., 1989).

Methods of transfecting mammalian cells and expressing DNA sequencesintroduced in the cells are described in e.g. Kaufman and Sharp, J. Mol.Biol 159 (1982), 601-621; Southern and Berg, J. Mol. Appl. Genet. 1(1982), 327-341; Loyter et al., Proc. Natl. Acad. Sci. USA 79 (1982),422-426; Wigler et al., Cell 14 (1978), 725; Corsaro and Pearson,Somatic Cell Genetics 7 (1981), 603, Graham and van der Eb, Virology 52(1973), 456; and Neumann et al., EMBO J. 1 (1982), 841-845.

Cloned DNA sequences are introduced into cultured mammalian cells by,for example, calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725-732,1978; Corsaro and Pearson, Somatic Cell Genetics7:603-616, 1981; Graham and Van der Eb, Virology 52d:456467, 1973) orelectroporation (Neumann et al., EMBO J. 1:841-845, 1982). To identifyand select cells that express the exogenous DNA, a gene that confers aselectable phenotype (a selectable marker) is generally introduced intocells along with the gene or cDNA of interest. Preferred selectablemarkers include genes that confer resistance to drugs such as neomycin,hygromycin, and methotrexate. The selectable marker may be anamplifiable selectable marker. A preferred amplifiable selectable markeris a dihydrofolate reductase (DHFR) sequence. Selectable markers arereviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers,Stoneham, Mass., incorporated herein by reference). The person skilledin the art will easily be able to choose suitable selectable markers.

Selectable markers may be introduced into the cell on a separate plasmidat the same time as the gene of interest, or they may be introduced onthe same plasmid. If, on the same plasmid, the selectable marker and thegene of interest may be under the control of different promoters or thesame promoter, the latter arrangement producing a dicistronic message.Constructs of this type are known in the art (for example, Levinson andSimonsen, U.S. Pat. No. 4,713,339). It may also be advantageous to addadditional DNA, known as “carrier DNA,” to the mixture that isintroduced into the cells.

After the cells have taken up the DNA, they are grown in an appropriategrowth medium, typically 1-2 days, to begin expressing the gene ofinterest. As used herein the term “appropriate growth medium” means amedium containing nutrients and other components required for the growthof cells and the expression of the human Factor VII polypeptide variantsof interest. Media generally include a carbon source, a nitrogen source,essential amino acids, essential sugars, vitamins, salts, phospholipids,protein and growth factors. For production of gamma-carboxylatedproteins, the medium will contain vitamin K, preferably at aconcentration of about 0.1 μg/ml to about 5 μg/ml. Drug selection isthen applied to select for the growth of cells that are expressing theselectable marker in a stable fashion. For cells that have beentransfected with an amplifiable selectable marker the drug concentrationmay be increased to select for an increased copy number of the clonedsequences, thereby increasing expression levels. Clones of stablytransfected cells are then screened for expression of the human FactorVII polypeptide variant of interest.

The host cell into which the DNA sequences encoding the human Factor VIIpolypeptide variants is introduced may be any cell, which is capable ofproducing the posttranslational modified human Factor VII polypeptidevariants and includes yeast, fungi and higher eucaryotic cells.

Examples of mammalian cell lines for use in the present invention arethe COS-1 (ATCC CRL 1650), baby hamster kidney (BHK) and 293 (ATCC CRL1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) cell lines. Apreferred BHK cell line is the tk⁻ ts13 BHK cell line (Waechter andBaserga, Proc. Natl. Acad. Sci. USA 79:1106-1110, 1982, incorporatedherein by reference), hereinafter referred to as BHK 570 cells. The BHK570 cell line has been deposited with the American Type CultureCollection, 12301 Parklawn Dr., Rockville, Md. 20852, under ATCCaccession number CRL 10314. A tk⁻ ts13 BHK cell line is also availablefrom the ATCC under accession number CRL 1632. In addition, a number ofother cell lines may be used within the present invention, including RatHep I (Rat hepatoma; ATCC CRL 1600), Rat Hep II (Rat hepatoma; ATCC CRL1548), TCMK (ATCC CCL 139), Human lung (ATCC HB 8065), NCTC 1469 (ATCCCCL 9.1), CHO (ATCC CCL 61) and DUKX cells (Urlaub and Chasin, Proc.Natl. Acad. Sci. USA 77:4216-4220,1980).

Examples of suitable yeasts cells include cells of Saccharomyces spp. orSchizosaccharomyces spp., in particular strains of Saccharomycescerevisiae or Saccharomyces kluyveri. Methods for transforming yeastcells with heterologous DNA and producing heterologous polypeptidesthere from are described, e.g. in U.S. Pat. No. 4,599,311, U.S. Pat. No.4,931,373, U.S. Pat. No. 4,870,008, 5,037,743, and U.S. Pat. No.4,845,075, all of which are hereby incorporated by reference.Transformed cells are selected by a phenotype determined by a selectablemarker, commonly drug resistance or the ability to grow in the absenceof a particular nutrient, e.g. leucine. A preferred vector for use inyeast is the POT 1 vector disclosed in U.S. Pat. No. 4,931,373. The DNAsequences encoding the human Factor VII polypeptide variants may bepreceded by a signal sequence and optionally a leader sequence, e.g. asdescribed above. Further examples of suitable yeast cells are strains ofKluyveromyces, such as K. lactis, Hansenula, e.g. H. polymorpha, orPichia, e.g. P. pastoris (cf. Gleeson et al., J. Gen. Microbiol.132,1986, pp. 3459-3465; U.S. Pat. No. 4,882,279).

Examples of other fungal cells are cells of filamentous fungi, e.g.Aspergillus spp., Neurospora spp., Fusarium 6 pp. or Trichoderma spp.,in particular strains of A. oryzae, A. nidulans or A. niger. The use ofAspergillus spp. for the expression of proteins is described in, e.g.,EP 272 277, EP 238 023, EP 184 438 The transformation of F. oxysporummay, for instance, be carried out as described by Malardier et al.,1989, Gene 78: 147-156. The transformation of Trichoderma spp. may beperformed for instance as described in EP 244 234.

When a filamentous fungus is used as the host cell, it may betransformed with the DNA construct of the invention, conveniently byintegrating the DNA construct in the host chromosome to obtain arecombinant host cell. This integration is generally considered to be anadvantage as the DNA sequence is more likely to be stably maintained inthe cell. Integration of the DNA constructs into the host chromosome maybe performed according to conventional methods, e.g. by homologous orheterologous recombination.

Transformation of insect cells and production of heterologouspolypeptides therein may be performed as described in U.S. Pat. No.4,745,051; U.S. Pat. No. 4,879,236; U.S. Pat. Nos. 5,155,037; 5,162,222;EP 397,485) all of which are incorporated herein by reference. Theinsect cell line used as the host may suitably be a Lepidoptera cellline, such as Spodoptera frugiperda cells or Trichoplusia ni cells (cf.U.S. Pat. No. 5,077,214). Culture conditions may suitably be asdescribed in, for instance, WO 89/01029 or WO 89/01028, or any of theaforementioned references.

The transformed or transfected host cell described above is thencultured in a suitable nutrient medium under conditions permittingexpression of the human Factor VII polypeptide variant after which allor part of the resulting peptide may be recovered from the culture. Themedium used to culture the cells may be any conventional medium suitablefor growing the host cells, such as minimal or complex media containingappropriate supplements. Suitable media are available from commercialsuppliers or may be prepared according to published recipes (e.g. incatalogues of the American Type Culture Collection). The human FactorVII polypeptide variant produced by the cells may then be recovered fromthe culture medium by, conventional procedures including separating thehost cells from the medium by centrifugation or filtration,precipitating the proteinaqueous components of the supernatant orfiltrate by means of a salt, e.g. ammonium sulphate, purification by avariety of chromatographic procedures, e.g. ion exchange chromatography,gelfiltration chromatography, affinity chromatography, or the like,dependent on the type of polypeptide in question.

Transgenic animal technology may be employed to produce the Factor VIIpolypeptide variants of the invention. It is preferred to produce theproteins within the mammary glands of a host female mammal. Expressionin the mammary gland and subsequent secretion of the protein of interestinto the milk overcomes many difficulties encountered in isolatingproteins from other sources. Milk is readily collected, available inlarge quantities, and biochemically well characterized. Furthermore, themajor milk proteins are present in milk at high concentrations(typically from about 1 to 15 g/l).

From a commercial point of view, it is clearly preferable to use as thehost a species that has a large milk yield. While smaller animals suchas mice and rats can be used (and are preferred at the proof ofprinciple stage), it is preferred to use livestock mammals including,but not limited to, pigs, goats, sheep and cattle. Sheep areparticularly preferred due to such factors as the previous history oftransgenesis in this species, milk yield, cost and the readyavailability of equipment for collecting sheep milk (see, for example,WO 88/00239 for a comparison of factors influencing the choice of hostspecies). It is generally desirable to select a breed of host animalthat has been bred for dairy use, such as East Friesland sheep, or tointroduce dairy stock by breeding of the transgenic line at a laterdate. In any event, animals of known, good health status should be used.

To obtain expression in the mammary gland, a transcription promoter froma milk protein gene is used. Milk protein genes include those genesencoding caseins (see U.S. Pat. No. 5,304,489), beta-lactoglobulin,a-lactalbumin, and whey acidic protein. The beta-lactoglobulin (BLG)promoter is preferred. In the case of the ovine beta-lactoglobulin gene,a region of at least the proximal 406 bp of 5′ flanking sequence of thegene will generally be used, although larger portions of the 5′ flankingsequence, up to about 5 kbp, are preferred, such as a −4.25 kbp DNAsegment encompassing the 5′ flanking promoter and non-coding portion ofthe beta-lactoglobulin gene (see Whitelaw et al., Biochem. J. 286: 31-39(1992)). Similar fragments of promoter DNA from other species are alsosuitable.

Other regions of the beta-lactoglobulin gene may also be incorporated inconstructs, as may genomic regions of the gene to be expressed. It isgenerally accepted in the art that constructs lacking introns, forexample, express poorly in comparison with those that contain such DNAsequences (see Brinster et al., Proc. Natl. Acad. Sci. USA 85: 836-840(1988); Palmiter et al., Proc. Natl. Acad. Sci. USA 88: 478-482 (1991);Whitelaw et al., Transgenic Res. 1: 3-13 (1991); WO 89/01343; and WO91/02318, each of which is incorporated herein by reference). In thisregard, it is generally preferred, where possible, to use genomicsequences containing all or some of the native introns of a geneencoding the protein or polypeptide of interest, thus the furtherinclusion of at least some introns from, e.g, the beta-lactoglobulingene, is preferred. One such region is a DNA segment that provides forintron splicing and RNA polyadenylation from the 3′ non-coding region ofthe ovine beta-lactoglobulin gene. When substituted for the natural 3′non-coding sequences of a gene, this ovine beta-lactoglobulin segmentcan both enhance and stabilize expression levels of the protein orpolypeptide of interest. Within other embodiments, the regionsurrounding the initiation ATG of the variant Factor VII sequence isreplaced with corresponding sequences from a milk specific protein gene.Such replacement provides a putative tissue-specific Initiationenvironment to enhance expression. It is convenient to replace theentire variant Factor VII pre-pro and 5 non-coding sequences with thoseof, for example, the BLG gene, although smaller regions may be replaced.

For expression of Factor VII polypeptide variants in transgenic animals,a DNA segment encoding variant Factor VII is operably linked toadditional DNA segments required for its expression to produceexpression units. Such additional segments include the above-mentionedpromoter, as well as sequences that provide for termination oftranscription and polyadenylation of mRNA. The expression units willfurther include a DNA segment encoding a secretory signal sequenceoperably linked to the segment encoding modified Factor VII. Thesecretory signal sequence may be a native Factor VII secretory signalsequence or may be that of another protein, such as a milk protein (see,for example, von Heijne, Nucl. Acids Res. 14: 4683-4690 (1986); andMeade et al., U.S. Pat. No. 4,873,316, which are incorporated herein byreference).

Construction of expression units for use in transgenic animals isconveniently carried out by inserting a variant Factor VII sequence intoa plasmid or phage vector containing the additional DNA segments,although the expression unit may be constructed by essentially anysequence of ligations. It is particularly convenient to provide a vectorcontaining a DNA segment encoding a milk protein and to replace thecoding sequence for the milk protein with that of a Factor VII variant;thereby creating a gene fusion that includes the expression controlsequences of the milk protein gene. In any event, cloning of theexpression units in plasmids or other vectors facilitates theamplification of the variant Factor VII sequence. Amplification isconveniently carried out in bacterial (e.g. E. coli) host cells, thusthe vectors will typically include an origin of replication and aselectable marker functional in bacterial host cells. The expressionunit is then introduced into fertilized eggs (including early-stageembryos) of the chosen host species. Introduction of heterologous DNAcan be accomplished by one of several routes, including microinjection(e.g. U.S. Pat. No. 4,873,191), retroviral infection (Jaenisch, Science240: 1468-1474 (1988)) or site-directed integration using embryonic stem(ES) cells (reviewed by Bradley et al., Bio/Technology 10: 534-539(1992)). The eggs are then implanted into the oviducts or uteri ofpseudopregnant females and allowed to develop to term. Offspringcarrying the introduced DNA in their germ line can pass the DNA on totheir progeny in the normal, Mendelian fashion, allowing the developmentof transgenic herds. General procedures for producing transgenic animalsare known in the art (see, for example, Hogan et al., Manipulating theMouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, 1986;Simons et al., Bio/Technology 6: 179-183 (1988); Wall et al., Biol.Reprod. 32: 645-651 (1985); Buhler et al., Bio/Technology 8: 140-143(1990); Ebert et al., Bio/Technology 9: 835-838 (1991); Krimpenfort etal., Bio/Technology 9: 844-847 (1991); Wall et al., J. Cell. Biochem.49: 113-120 (1992); U.S. Pat. No. 4,873,191; U.S. Pat. No. 4,873,316; WO88/00239, WO 90/05188, WO 92111757; and GB 87100458). Techniques forintroducing foreign DNA sequences into mammals and their germ cells wereoriginally developed in the mouse (see, e.g., Gordon et al., Proc. Natl.Acad. Sci. USA 77: 7380-7384 (1980); Gordon and Ruddle, Science 214:1244-1246 (1981); Palmiter and Brinster, Cell 41: 343-345 (1985);Brinster et al., Proc. Natl. Acad. Sci. USA 82: 4438-4442 (1985); andHogan et al. (ibid.)). These techniques were subsequently adapted foruse with larger animals, including livestock species (see, e.g., WO88/00239, WO 90/05188, and WO 92/11757; and Simons et al.,Bio/Technology 6:179-183 (1988)). To summarise, in the most efficientroute used to date in the generation of transgenic mice or livestock,several hundred linear molecules of the DNA of interest are injectedinto one of the pro-nuclei of a fertilized egg according to establishedtechniques. Injection of DNA into the cytoplasm of a zygote can also beemployed.

Production in transgenic plants may also be employed. Expression may begeneralised or directed to a particular organ, such as a tuber (see,Hiatt, Nature 344:469-479 (1990); Edelbaum et al., J. Interferon Res.12:449-453 (1992); Sijmons et al., Bio/Technology 8:217-221 (1990); andEP 0 255 378).

The Factor VII polypeptide variants of the invention are recovered fromcell culture medium or milk. The Factor VII polypeptide variants of thepresent invention may be purified by a variety of procedures known inthe art including, but not limited to, chromatography (e.g., ionexchange, affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing(IEF), differential solubility (e.g., ammonium sulfate precipitation),or extraction (see, e.g., Protein Purification, J.-C. Janson and LarsRyden, editors, VCH Publishers, New York, 1989). Preferably, they may bepurified by affinity chromatography on an anti-Factor VII antibodycolumn. The use of calcium-dependent monoclonal antibodies, as describedby Wakabayashi et al., J. Biol. Chem. 261:11097-11108, (1986) and Thimet al., Biochemistry 27: 7785-7793, (1988), is particularly preferred.Additional purification may be achieved by conventional chemicalpurification means, such as high performance liquid chromatography.Other methods of purification, including barium citrate precipitation,are known in the art, and may be applied to the purification of thenovel Factor VII polypeptide variants described herein (see, forexample, Scopes, R., Protein Purification, Springer-Verlag, N.Y., 1982).

For therapeutic purposes it is preferred that the Factor VII polypeptidevariants of the invention are substantially pure. Thus, in a preferredembodiment of the invention the Factor VII polypeptide variants of theinvention is purified to at least about 90 to 95% homogeneity,preferably to at least about 98% homogeneity. Purity may be assessed bye.g. gel electrophoresis and amino-terminal amino acid sequencing.

The Factor VII variant is cleaved at its activation site in order toconvert it to its two-chain form. Activation may be carried outaccording to procedures known in the art, such as those disclosed byOsterud, et al., Biochemistry 11:2853-2857 (1972); Thomas, U.S. Pat. No.4,456,591; Hedner and Kisiel, J. Clin. Invest. 71:1836-1841 (1983); orKisiel and Fujikawa, Behring Inst. Mitt. 73:29-42 (1983). Alternatively,as described by Bjorn et al. (Research Disclosure, 269 September 1986,pp. 564-565), Factor VII may be activated by passing it through anion-exchange chromatography column, such as Mono Q® (Pharmacia fineChemicals) or the like. The resulting activated Factor VII variant maythen be formulated and administered as described below.

ASSAYS

The invention also provides suitable assays for selecting preferredFactor VII variants according to the invention. These assays can beperformed as a simple preliminary in vitro test.

Thus, Example 3 herein discloses a simple test (entitled “In VitroHydrolysis Assay”) for the activity of Factor VII variants of theinvention. Based thereon, Factor VIIa variants which are of particularinterest are such variants where the ratio between the activity of thevariant and the activity of native Factor VII shown in FIG. 1 is above1.0, e.g. at least about 1.25, preferably at least about 2.0, such as atleast about 3.0 or, even more preferred, at least about 4.0 when testedin the “In Vitro Hydrolysis Assay”.

The activity of the variants can also be measured using a physiologicalsubstrate such as factor X (“In Vitro Proteolysis Assay”) (see Example4), suitably at a concentration of 100-1000 nM, where the factor Xagenerated is measured after the addition of a suitable chromogenicsubstrate (eg. S-2765). In addition, the activity assay may be run atphysiological temperature.

The ability of the Factor VIIa variants to generate thrombin can also bemeasured in an assay comprising all relevant coagulation factors andinhibitors at physiological concentrations (minus factor VIII whenmimicking hemophilia A conditions) and activated platelets (as describedon p. 543 in Monroe et al. (1997) Brit. J. Haematol. 99, 542-547 whichis hereby incorporated as reference).

Administration and pharmaceutical compositions The Factor VIIpolypeptide variants according to the present invention may be used tocontrol bleeding disorders which have several causes such as clottingfactor deficiencies (e.g. haemophilia A and B or deficiency ofcoagulation factors XI or VII) or clotting factor inhibitors, or theymay be used to control excessive bleeding occurring in subjects with anormally functioning blood clotting cascade (no clotting factordeficiencies or inhibitors against any of the coagulation factors). Thebleedings may be caused by a defective platelet function,thrombocytopenia or von Willebrand's disease. They may also be seen insubjects in whom an increased fibrinolytic activity has been induced byvarious stimuli.

In subjects who experience extensive tissue damage in association withsurgery or vast trauma, the haemostatic mechanism may be overwhelmed bythe demand, of immediate haemostasis and they may develop bleedings inspite of a normal haemostatic mechanism. Achieving satisfactoryhaemostasis is also a problem when bleedings occur in organs such as thebrain, inner ear region and eyes and may also be a problem in cases ofdiffuse bleedings (haemorrhagic gastritis and profuse uterine bleeding)when it is difficult to identify the source. The same problem may arisein the process of taking biopsies from various organs (liver, lung,tumour tissue, gastrointestinal tract) as well as in laparoscopicsurgery. These situations share the difficulty of providing haemostasisby surgical techniques (sutures, clips, etc.). Acute and profusebleedings may also occur in subjects on anticoagulant therapy in whom adefective haemostasis has been induced by the therapy given. Suchsubjects may need surgical interventions in case the anticoagulanteffect has to be counteracted rapidly. Another situation that may causeproblems in the case of unsatisfactory haemostasis is when subjects witha normal haemostatic mechanism are given anticoagulant therapy toprevent thromboembolic disease. Such therapy may include heparin, otherforms of proteoglycans, warfarin or other forms of vitamin K-antagonistsas well as aspirin and other platelet aggregation inhibitors.

A systemic activation of the coagulation cascade may lead todisseminated intravascular coagulation (DIC). However, suchcomplications have not been seen in subjects treated with high doses ofrecombinant Factor VIIa because of a localised haemostatic process ofthe kind induced by the complex formation between Factor VIIa and TFexposed at the site of vessel wall injury. The Factor VII polypeptidevariants according to the invention may thus also be used in theiractivated form to control such excessive bleedings associated with anormal haemostatic mechanism.

For treatment in connection with deliberate interventions, the FactorVII polypeptide variants of the invention will typically be administeredwithin about 24 hours prior to performing the intervention, and for asmuch as 7 days or more thereafter. Administration as a coagulant can beby a variety of routes as described herein.

The dose of the Factor VII polypeptide variants ranges from about 0.05mg to 500 mg/day, preferably from about 1 mg to 200 mg/day, and morepreferably from about 10 mg to about 175 mg/day for a 70 kg subject asloading and maintenance doses, depending on the weight of the subjectand the severity of the condition.

The pharmaceutical compositions are primarily intended for parenteraladministration for prophylactic and/or therapeutic treatment.Preferably, the pharmaceutical compositions are administeredparenterally, i.e., intravenously, subcutaneously, or intramuscularly,or it may be administered by continuous or pulsatile infusion. Thecompositions for parenteral administration comprise the Factor VIIvariant of the invention in combination with, preferably dissolved in, apharmaceutically acceptable carrier, preferably an aqueous carrier. Avariety of aqueous carriers may be used, such as water, buffered water,0.4% saline, 0.3% glycine and the like. The Factor VII polypeptidevariants of the invention can also be formulated into liposomepreparations for delivery or targeting to the sites of injury. Liposomepreparations are generally described in, e.g., U.S. Pat. No. 4,837,028,U.S. Pat. No. 4,501,728, and U.S. Pat. No. 4,975,282. The compositionsmay be sterilised by conventional, well-known sterilisation techniques.The resulting aqueous solutions may be packaged for use or filteredunder aseptic conditions and lyophilised, the lyophilised preparationbeing combined with a sterile aqueous solution prior to administration.The compositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH adjusting and buffering agents, tonicity adjusting agents and thelike, for example, sodium acetate, sodium lactate, sodium chloride,potassium chloride, calcium chloride, etc.

The concentration of Factor VII variant in these formulations can varywidely, i.e., from less than about 0.5% by weight, usually at or atleast about 1% by weight to as much as 15 or 20% by weight and will beselected primarily by fluid volumes, viscosities, etc., in accordancewith the particular mode of administration selected.

Thus, a typical pharmaceutical composition for intravenous infusion canbe made up to contain 250 ml of sterile Ringer's solution and 10 mg ofthe Factor VII variant. Actual methods for preparing parenterallyadministrable compositions will be known or apparent to those skilled inthe art and are described in more detail in, for example, Remington'sPharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, Pa.(1990).

The compositions containing the Factor VII polypeptide variants of thepresent invention can be administered for prophylactic and/ortherapeutic treatments. In therapeutic applications, compositions areadministered to a subject already suffering from a disease, as describedabove, in an amount sufficient to cure, alleviate or partially arrestthe disease and its complications. An amount adequate to accomplish thisis defined as “therapeutically effective amount”. As will be understoodby the person skilled in the art amounts effective for this purpose willdepend on the severity of the disease or injury as well as the weightand general state of the subject. In general, however, the effectiveamount will range from about 0.05 mg up to about 500 mg of the FactorVII variant per day for a 70 kg subject, with dosages of from about 1.0mg to about 200 mg of the Factor VII variant per day being more commonlyused.

The FVIIa polypeptides of the present invention may generally beemployed in serious disease or injury states, that is, life threateningor potentially life threatening situations. In such cases, in view ofthe minimisation of extraneous substances and general lack ofimmunogenicity of human Factor VII polypeptide variants in humans, itmay be felt desirable by the treating physician to administer asubstantial excess of these variant Factor VII compositions.

In prophylactic applications, compositions containing the Factor VIIvariant of the invention are administered to a subject susceptible to orotherwise at risk of a disease state or injury to enhance the subject'sown coagulative capability. Such an amount is defined to be a“prophylactically effective dose.” In prophylactic applications, theprecise amounts once again depend on the subject's state of health andweight, but the dose generally ranges from about 0.05 mg to about 500 mgper day for a 70-kilogram subject, more commonly from about 1.0 mg toabout 200 mg per day for a 70-kilogram subject.

Single or multiple administrations of the compositions can be carriedout with dose levels and patterns being selected by the treatingphysician. For ambulatory subjects requiring daily maintenance levels,the Factor VII polypeptide variants may be administered by continuousinfusion using e.g. a portable pump system.

Local delivery of the Factor VII variant of the present invention, suchas, for example, topical application may be carried out, for example, bymeans of a spray, perfusion, double balloon catheters, stent,incorporated into vascular grafts or stents, hydrogels used to coatballoon catheters, or other well established methods. In any event, thepharmaceutical compositions should provide a quantity of Factor VIIvariant sufficient to effectively treat the subject.

The present invention is further illustrated by the following exampleswhich, however, are not to be construed as limiting the scope ofprotection. The features disclosed in the foregoing description and inthe following examples may, both separately and in any combinationthereof, be material for realising the invention in diverse formsthereof.

EXAMPLES

The terminology for amino acid substitutions used in the followingexamples are as follows. The first letter represent the amino acidnaturally present at a position of SEQ ID NO:1. The following numberrepresent the position in SEQ ID NO:1. The second letter represent thedifferent amino acid substituting for the natural amino acid. An exampleis L305V/F374Y-FVII, the leucine at position 305 of SEQ ID NO:1 isreplaced by a valine and the phenylalanine at position 374 of SEQ IDNO:1 is replaced by an tyrosine, both mutations in the same Factor VIIvariant.

Example 1

DNA encoding L305V/S314E/F374Y-FVII, L305V/K337A/F374Y-FVII,L305V/S314E/K337A/F374Y-F-VII, andV158D/E296V/M298Q/L305V/S314E/K337A/F374Y-FVII.

DNA constructs encoding L305V/S314E/F374Y-FVII, L305V/K337A/F374Y-FVII,L305V/S314E/K337A/F374Y-FVII were prepared by site-directed mutagenesisusing a super-coiled, double stranded DNA vector with an insert ofinterest and two synthetic primers containing the desired mutation. Thefollowing primers were used (in subsequent PCR reactions if necessary toobtain the desired FVII variant):

For L305V-FVII:

5′-CGT GCC CCG GGT GAT GAC CCA GGA (SEQ ID NO: 2) C-3′ 5′-GTC CTG GGTCAT CAC CCG GGG CAC (SEQ ID NO: 3) G-3′For K337A-FVII:

5′-CGG ATG GCA GCG CGG ACT CCT GCA (SEQ ID NO: 4) AGG G-3′ 5′-CCC TTGCAG GAG TCC GCG CTG CCA (SEQ ID NO: 5) TCC G-3′For V156D-FVII:

5′-GTG GGG GGC AAG GAC TGC CCC AAA (SEQ ID NO: 6) GGG G-3′ 5′-CCC CTTTGG GGC AGT CCT TGC CCC (SEQ ID NO: 7) CCA C-3′

For E296V/M298Q-FVII:

5′-GCC ACG GCC CTG GTG CTC CAG GTC (SEQ ID NO: 8) CTC AAC GTG CCC-3′5′-GGG CAC GTT GAG GAC CTG GAG CAC (SEQ ID NO: 9) CAG GGC CGT GGC-3′

-   For S314E-FVII

5′-GCC TGC AGC AGG AAC GGA AGG TGG (SEQ ID NO: 10) GAG ACT CC-3′ 5′-GGAGTC TCC CAC CTT CCG TTC CTG (SEQ ID NO: 11) CTG GAG GC-3′For F374Y-F VII:

5′-CGC AAC CGT GGG CCA CTA TGG GGT (SEQ ID NO: 12) GTA CAC C-3′ 5′-GGTGTA CAC CCC ATA GTG GCC CAC (SEQ ID NO: 13) GGT TGC G-3′

The oligonucleotide primers, each complementary to opposite strands ofthe vector, were extended during temperature cycling by means of Pfu DNApolymerase. On incorporation of the primers, a mutated plasmidcontaining staggered nicks was generated. Following temperature cycling,the product was treated with Dpnl which is specific for methylated andhemi-methylated DNA to digest the parental DNA template and to selectfor mutation-containing synthesized DNA.

Procedures for preparing a DNA construct for all the FVII variantsaccording to the present invention using polymerase chain reaction usingspecific primers to achieve the desire FVII variant are well known topersons skilled in the art (cf. PCR Protocols, 1990, Academic Press, SanDiego, Calif., USA).

V158D/E296V/M298Q/L305V/S314E/K337A/F374Y-FVII can be prepared by thesame method.

Example 2

Preparation of L305V/S314E/F374Y-FVII, L305V/K337A/F374Y-FVII, andL305V/S314/K337A/F374Y-FVII.

BHK cells we re transfected essentially as previously described (Thim etal. (1988) Biochemistry 27, 77857793; Persson and Nielsen (1996) FEBSLeft. 385, 241243) to obtain expression of the specific FVII variant. The Factor VII variant was purified as follows:

Conditioned medium was loaded onto a 25-ml column of Q Sepharose FastFlow (Pharmacia Biotech) after addition of 5 mM EDTA, 0.1% Triton X-100and 10 mM Tris, adjustment of pH to 8.0 and adjust ment of th econductivity to 10-11 mS/cm by adding water.

Elution of the protein was accomplished by stepping from 10 mM Tris, 50mM NaCl, 0.1% Triton X-100, pH 8.0 to 10 mM Tris, 50 mM NaCl, 25 mMCaCl₂, 0.1% Triton X-100, pH 8.0. The fractions containing the specificFVII variant we re pooled a nd applied to a 25-ml column containingmonoclonal antibody F1A2 (Novo Nordisk, Bagsvaerd, Denmark) coupled toCNBr-activated Sepharose 4B (Pharmacia Biotech).

The column was equilibrated with 50 mM Hepes, pH 7.5, containing 10 mMCaCl₂,100 mM NaCl and 0.02% Triton X-100. After washing withequilibration buffer and equilibration buffer containing 2 M NaCl, boundmaterial was eluted with equilibration buffer containing 10 mM EDTAinstead of CaCl₂. Before use or storage, excess CaCl₂ over EDTA wasadded or the specific FVII variant was transferred to a Ca²⁺-containingbuffer. The yield of each step was followed by factor VII ELISAmeasurements and the purified protein was analysed by SDS-PAGE. Othervariants FVII variants according to example 1 and all other variantsaccording to the present invention may be prepared by the same method.

Example 3

In Vitro Hydrolysis Assay

Native (wild-type) Factor VIIa and Factor VIIa variant (both hereafterreferred to as “Factor VIIa”) were assayed in parallel to directlycompare their specific activities. The assay was carried out in amicrotiter plate (MaxiSorp, Nunc, Denmark). The chromogenic substrateD-Ile-Pro-Arg-p-nitroanilide (S-2288, Chromogenix, Sweden), finalconcentration 1 mM, was added to Factor VIIa (final concentration 100nM) In 50 mM Hepes, pH 7.4, containing 0.1 M NaCl, 5 mM CaCl₂ and 1mg/ml bovine serum albumin. The absorbance at 405 nm was measuredcontinuously in a SpectraMax™ 340 plate reader (Molecular Devices, USA).The absorbance developed during a 20 minute incubation, aftersubtraction of the absorbance in a blank well containing no enzyme, wasused to calculate the ratio between the activities of variant andwild-type Factor VIIa:Ratio=(A _(405 nm)Factor VIIa variant)/(A _(405 nm) Factor VIIawild-type).

Example 4

In Vitro Proteolysis Assay

Native (wild-type) Factor VIIa and Factor VII variant (both hereafterreferred to as “Factor VIIa”) were assayed in parallel to directlycompare their specific activities. The assay was carried out in amicrotiter plate (MaxiSorp, Nunc, Denmark). Factor VIIa (10 nM) andFactor X (0.8 microM) in 100 microL 50 mM Hepes, pH 7.4, containing 0.1M NaCl, 5 mM CaCl₂ and 1 mg/ml bovine serum albumin, are incubated for15 min. Factor X cleavage was then stopped by the addition of 50 microL50 mM Hepes, pH 7.4, containing 0.1 M NaCl, 20mM EDTA and 1 mg/ml bovineserum albumin. The amount of Factor Xa generated was measured byaddition of the chromogenic substrate Z-D-Arg-Gly-Arg-p-nitroanilide(S-2765, Chromogenix, Sweden), final concentration 0.5 mM. Theabsorbance at 405 nm was measured continuously in a SpectraMax™ 340plate reader (Molecular Devices, USA). The absorbance developed during10 minutes, after subtraction of the absorbance in a blank wellcontaining no FVIIa, was used to calculate the ratio between theproteolytic activities of variant and wild-type Factor VIIa:Ratio=(A _(405 nm) Factor VIIa variant)/(A _(405 nm) Factor VIIawild-type).

Example 5

Relative Activities of FVIIa Variants Measured in the Assays Describedin Examples 3 AND 4.

Ratio Variant in example 3 Ratio in example 4 L305V/S314E/F374Y-FVlla 33 L305V/K337A/F374Y-FVlla 12 8 L305V/S314E/K337A/F374Y-FVlla 23 11wt-FVlla 1.0 1.0

1. A Factor VII polypeptide comprising at least two substitutionsrelative to the amino acid sequence of SEQ ID NO:1, wherein saidsubstitutions comprise: (i) a first substitution consisting ofreplacement of F374 with any other amino acid, and (ii) one or moresubstitutions consisting of replacement with any other amino acid of oneor more amino acids selected from the group consisting of L305, S314,K157, K337, D334, S336, V158, E296, and M298.
 2. The Factor VIIpolypeptide according to claim 1, wherein F374 is replaced with anyother amino acid and K157 is replaced with any other amino acid.
 3. TheFactor VII polypeptide according to claim 1, wherein F374 is replacedwith any other amino acid and K337 is replaced with any other aminoacid.
 4. The Factor VII polypeptide according to claim 1, wherein F374is replaced with any other amino acid and D334 is replaced with anyother amino acid.
 5. The Factor VII polypeptide according to claim 1,wherein F374 is replaced with any other amino acid and S336 is replacedwith any other amino acid.
 6. The Factor VII polypeptide according toclaim 1, wherein F374 is replaced with any other amino acid and V158 isreplaced with any other amino acid.
 7. The Factor VII polypeptideaccording to claim 1, wherein F374 is replaced with any other amino acidand E296 Is replaced with any other amino acid.
 8. The Factor VIIpolypeptide according to claim 1, wherein F374 is replaced with anyother amino acid and M298 is replaced with any other amino acid.
 9. TheFactor VII polypeptide according to claim 1, wherein F374 is replacedwith any other amino acid and L305 is replaced with any other aminoacid.
 10. The Factor VII polypeptide according to claim 1, wherein F374is replaced with any other amino acid and S314 is replaced with anyother amino acid.
 11. The Factor VII polypeptide according to claim 1,further comprising at least one additional substitution at an amino acidresidue in the protease domain, wherein said residue has been replacedwith any other amino acid, and wherein the ratio between the activity ofsaid Factor VII polypeptide and the activity of the native Factor VIIapolypeptide shown in SEQ ID NO:1 is at least 1.25.
 12. The Factor VIIpolypeptide according to claim 11, wherein at the most 20 additionalamino acids in the remaining positions in the protease domain have beenreplaced with any other amino acid.
 13. The Factor VII polypeptideaccording to claim 11, wherein the additional substitution is at aresidue corresponding to a position selected from 159-170 of SEQ IDNO:1.
 14. The Factor VII polypeptide according to claim 11, wherein theadditional substitution is at a residue corresponding to a positionselected from 290-304 of SEQ ID NO:1.
 15. The Factor VII polypeptideaccording to claim 14, wherein R304 has been replaced by an amino acidselected from the group consisting of Tyr, Phe, Leu, and Met.
 16. TheFactor VII polypeptide according to claim 11, wherein the additionalsubstitution is at a residue corresponding to a position selected from306-312 of SEQ ID NO:1.
 17. The Factor VII polypeptide according toclaim 16, wherein M306 has been replaced by an amino acid selected fromthe group consisting of Asp and Asn.
 18. The Factor VII polypeptideaccording to claim 16, wherein D309 has been replaced by an amino acidselected from the group consisting of Ser and Thr.
 19. The Factor VIIpolypeptide according to claim 11, wherein the additional substitutionis at a residue corresponding to a position selected from 330-339 of SEQID NO:1.
 20. The Factor VII polypeptide according to claim 11, whereinA274 has been replaced with any other amino acid.
 21. The Factor VIIpolypeptide according to claim 20, wherein said A274 has been replacedwith an amino acid selected from the group consisting of Met, Leu, Lys,and Arg.
 22. The Factor VII polypeptide according to claim 1, whereinsaid K157 has been replaced by an amino acid selected from the groupconsisting of Gly, Val, Ser, Thr, Asn, Gln, Asp, and Glu.
 23. The FactorVII polypeptide according to claim 1, wherein said K337 has beenreplaced by an amino acid selected from the group consisting of Ala,Gly, Val, Ser, Thr, Asn, Gln, Asp, and Glu.
 24. The Factor VIIpolypeptide according to claim 1, wherein said D334 has been replaced byan amino acid selected from the group consisting of Gly and Glu.
 25. TheFactor VII polypeptide according to claim 1, wherein said S336 has beenreplaced by an amino acid selected from the group consisting of Gly andGlu.
 26. The Factor VII polypeptide according to claim 1, wherein saidV168 has been replaced by an amino acid selected from the groupconsisting of Ser, Thr, Asn, Gln, Asp, and Glu.
 27. The Factor VIIpolypeptide according to claim 1, wherein said E296 has been replaced byan amino acid selected from the group consisting of Arg, Lys, Ile, Leuand Val.
 28. The Factor VII polypeptide according to claim 1, whereinsaid M298 has been replaced by an amino acid selected from the groupconsisting of Lys, Arg, Gln, and Asn.
 29. The Factor VII polypeptideaccording to claim 1, wherein said L305 has been replaced by an aminoacid selected from the group consisting of Val, Tyr and Ile.
 30. TheFactor VII polypeptide according to claim 1, wherein said S314 has beenreplaced by an amino acid selected from the group consisting of Gly,Lys, Gln, and Glu.
 31. The Factor VII polypeptide according to claim 1,wherein said F374 has been replaced by an amino acid selected from thegroup consisting of Pro and Tyr.
 32. The Factor VII polypeptideaccording to claim 31, wherein said F374 has been replaced by Tyr. 33.The Factor VII polypeptide according to claim 1, wherein said Factor VIIpolypeptide is human Factor VII.
 34. The Factor VII polypeptideaccording to claim 1; wherein the ratio between the activity of saidFactor VII polypeptide and the activity of the native Factor VIIapolypeptide shown in SEQ ID NO:1 is at least 1.25.
 35. The Factor VIIpolypeptide according tol, which is F374Y/L305V-FVII.
 36. The Factor VIIpolypeptide according to claim 1, which is F374Y/L305V/S314E/K337A-FVII.37. The Factor VII polypeptide according to claim 1, which isF374Y/L305V/S314E-FVII.
 38. The Factor VII polypeptide according toclaim 1, which is F374Y/L305V/K337A-FVII.
 39. A method for the treatmentof bleeding disorders or bleeding episodes in a subject or for theenhancement of the normal haemostatic system, the method comprisingadministering a therapeutically or prophylactically effective amount ofa Factor VII polypeptide comprising at least two substitutions relativeto the amino acid sequence of SEQ ID NO:1, wherein said substitutionsare (i) replacement of F374 with any other amino acid, and (ii)replacement with any other amino acid of one or more amino acidsselected from the group consisting of L305, S314, K157, K337, D334,S336, V158, E296, and M298; to a subject in need thereof.